CTLA4-Cy4 fusion proteins

ABSTRACT

CTLA4-immunoglobulin fusion proteins having modified immunoglobulin constant region-mediated effector functions, and nucleic acids encoding the fusion proteins, are described. The CTLA4-immunoglobulin fusion proteins comprise two components: a first peptide having a CTLA4 activity and a second peptide comprising an immunoglobulin constant region which is modified to reduce at least one constant region-mediated biological effector function relative to a CTLA4-IgG1 fusion protein. The nucleic acids of the invention can be integrated into various expression vectors, which in turn can direct the synthesis of the corresponding proteins in a variety of hosts, particularly eukaryotic cells. The CTLA4-immunoglobulin fusion proteins described herein can be administered to a subject to inhibit an interaction between a CTLA4 ligand (e.g., B7-1 and/or B7-2) on an antigen presenting cell and a receptor for the CTLA4 ligand (e.g., CD28 and/or CTLA4) on the surface of T cells to thereby suppress an immune response in the subject, for example to inhibit transplantation rejection, graft versus host disease or autoimmune responses.

BACKGROUND OF THE INVENTION

To induce antigen-specific T cell activation and clonal expansion, twosignals provided by antigen-presenting cells (APCs) must be delivered tothe surface of resting T lymphocytes (Jenkins, M. and Schwartz, R.(1987) J. Exp. Med. 165:302-319; Mueller, D. L., et al. (1990) J.Immmunol. 144:3701-3709; Williams, I. R. and Unanue, E. R. (1990) J.Immunol. 145:85-93). The first signal, which confers specificity to theimmune response, is mediated via the T cell receptor (TCR) followingrecognition of foreign antigenic peptide presented in the context of themajor histocompatibility complex (MHC). The second signal, termedcostimulation, induces T cells to proliferate and become functional(Schwartz, R. H. (1990) Science 248:1349-1356). Costimulation is neitherantigen-specific, nor MHC restricted and is thought to be provided byone or more distinct cell surface molecules expressed by APCs (Jenkins,M. K., et al. (1988) J. Immunol. 140:3324-3330; Linsley, P. S., et al.(1991) J. Exp. Med. 173:721-730; Gimmi, C. D. et al., (1991) Proc. Natl.Acad. Sci. USA, 88:6575-6579; Young, J. W., et al. (1992) J. Clin.Invest. 90:229-237; Koulova, L., et al. (1991) J. Exp. Med. 173:759-762;Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA. 89:271-275;van-Seventer G. A., et al. (1990) J. Immunol. 144:4579-4586; LaSalle, J.M., et al., (1991) J. Immunol. 147:774-80; Dustin, M. I. et al., (1989)J. Exp. Med. 169:503; Armitage, R. J., et al. (1992) Nature 357:80-82;Liu, Y., et al. (1992) J. Exp. Med. 175:437-445).

Considerable evidence suggests that the B7-1 protein (CD80; originallytermed B7), expressed on APCs, is one such critical costimulatorymolecule (Linsley, P. S., et al., (1991) J. Exp. Med. 173:721-730;Gimmi, C. D., et al., (1991) Proc. Natl. Acad. Sci. USA. 88:6575-6579;Koulova, L., et al., (1991) J. Exp. Med. 173:759-762; Reiser, H., et al.(1992) Proc. Natl. Acad. Sci. USA, 89:271-275; Linsley, P. S. et al.(1990) Proc. Natl. Acad. Sci. USA. 87: 5031-5035; Freeman, G. J. et al.(1991) J. Exp. Med. 174:625-631.). Recent evidence suggests the presenceof additional costimulatory molecules on the surface of activated Blymphocytes (Boussiotis V. A., et al. (1993) Proc. Natl. Acad. Sci. USA.90:11059-11063; Freeman G. J., et al. (1993) Science 262:907-909;Freeman G. J., et al. (1993) Science 262:909-911; and Hathcock K. S., etal. (1993) Science 262:905-907). The human B lymphocyte antigen B7-2(CD86) has been cloned and is expressed by human B cells at about 24hours following stimulation with either anti-immunoglobulin or anti-MHCclass II monoclonal antibody (Freeman G. J., et al. (1993) Science262:909-911). At about 48 to 72 hours post activation, human B cellsexpress both B7-1 and a third CTLA4 counter-receptor which is identifiedby a monoclonal antibody BB-1, which also binds B7-1 (Yokochi, T., etal. (1982) J. Immunol. 128:823-827). The BB-1 antigen is also expressedon B7-1 negative activated B cells and can costimulate T cellproliferation without detectable IL-2 production, indicating that theB7-1 and BB-1 molecules are distinct (Boussiotis V. A., et al. (1993)Proc. Natl. Acad. Sci. USA 90:11059-11063). The presence of thesecostimulatory molecules on the surface of activated B lymphocytesindicates that T cell costimulation is regulated, in part, by thetemporal expression of these molecules following B cell activation.

B7-1 is a counter-receptor for two ligands expressed on T lymphocytes.The first ligand, termed CD28, is constitutively expressed on resting Tcells and increases after activation. After signaling through the T cellreceptor, ligation of CD28 induces T cells to proliferate and secreteIL-2 (Linsley, P. S., et al. (1991) J. Exp. Med. 173: 721-730; Gimmi, C.D., et al. (1991) Proc. Natl. Acad. Sci. USA. 88:6575-6579; Thompson, C.B., et al. (1989) Proc. Natl. Acad. Sci. USA. 86:1333-1337; June, C. H.,et al. (1990) Immunol. Today, 11:211-6; Harding, F. A., et al. (1992)Nature, 356:607-609.). The second ligand, termed CTLA4, is homologous toCD28, but is not expressed on resting T cells and appears following Tcell activation (Brunet, J. F., et al. (1987) Nature 328:267-270). LikeB7-1, B7-2 is a counter-receptor for both CD28 and CTLA4 (Freeman G. J.,et al. (1993) Science 262:909-911). CTLA4 was first identified as amouse cDNA clone, in a library of cDNA from a cytotoxic T cell clonesubtracted with RNA from a B cell lymphoma (Brunet, J. F., et al. (1987)supra). The mouse CTLA4 cDNA was then used as a probe to identify thehuman and mouse CTLA4 genes (Harper, K., et al. (1991) J. Immunol.147:1037-1044; and Dariavich, et al. (1988) Eur. J. Immunol.18(12):1901-1905, sequence modified by Linsley, P. S., et al. (1991) J.Exp. Med. 174:561-569). A probe from the V domain of the human gene wasused to detect the human cDNA which allowed the identification of theCTLA4 leader sequence (Harper, K., et al. (1991) supra).

Soluble derivatives of cell surface glycoproteins in the immunoglobulingene superfamily have been made consisting of an extracellular domain ofthe cell surface glycoprotein fused to an immunoglobulin constant (Fc)region (see e.g., Capon, D. J. et al. (1989) Nature 337:525-531 andCapon U.S. Pat. Nos. 5,116,964 and 5,428,130 [CD4-IgG1 constructs]:Linsley, P. S. et al. (1991) J. Exp. Med. 173:721-730 [a CD28-IgG1construct and a B7-1-IgG1 construct]: and Linsley, P. S. et al. (1991)J. Exp. Med. 174:561-569 and U.S. Pat. No. 5,434,131[a CTLA4-IgG1]).Such fusion proteins have proven useful for studying receptor-ligandinteractions. For example, a CTLA4-IgG immunoglobulin fusion protein wasused to study interactions between CTLA4 and its natural ligands(Linsley, P. S., et al., (1991) J. Exp. Med. 174:561-569; InternationalApplication WO93/00431; and Freeman G. J., et al. (1993) Science262:909-911).

The importance of the B7:CD28/CTLA4 costimulatory pathway has beendemonstrated in vitro and in several in vivo model systems. Blockade ofthis costimulatory pathway results in the development of antigenspecific tolerance in murine and human systems (Harding, F. A., et al.(1992) Nature 356:607-609; Lenschow, D. J., et al. (1992) Science257:789-792; Turka, L. A., et al. (1992) Proc. Natl. Acad. Sci. USA89:11102-11105; Gimmi, C. D., et al. (1993) Proc. Natl. Acad. Sci. USA90:6586-6590; Boussiotis, V., et al. (1993) J. Exp. Med. 178:1753-1763).Conversely, transfection of a B7-1 gene into B7-1 negative murine tumorcells to thereby express B7-1 protein on the tumor cell surface inducesT-cell mediated specific immunity accompanied by tumor rejection andlong lasting protection to tumor challenge (Chen, L., et al. (1992) Cell71:1093-1102; Townsend, S. E. and Allison, J. P. (1993) Science259:368-370; Baskar, S., et al. (1993) Proc. Natl. Acad. Sci. USA90:5687-5690.). Therefore, approaches which manipulate the B7:CD28/CTLA4interaction to thereby stimulate or suppress immune responses would bebeneficial therapeutically.

SUMMARY OF THE INVENTION

This invention pertains to CTLA4-immunoglobulin fusion proteins havingmodified immunoglobulin constant (IgC) region-mediated effectorfunctions and to nucleic acids encoding the proteins. In one embodiment,the fusion proteins of the present invention have been constructed byfusing a peptide having a CTLA4 activity and a second peptide comprisingan immunoglobulin constant region to create a CTLA4Ig fusion protein. Inanother embodiment, the variable regions of immunoglobulin heavy andlight chains have been replaced by the B7-binding extracellular domainof CTLA4 to create CTLA4-Ab fusion proteins. As used herein, the term“CTLA4-immunoglobulin fusion protein” refers to both the CTLA4Ig andCTLA4-Ab forms. In a preferred embodiment, the fusion proteins of theinvention have been modified to reduce their ability to activatecomplement and/or bind to Fc receptors. In one embodiment, an IgC regionof an isotype other than Cγ1 is used in the fusion protein and themodified effector function(s) can be assessed relative to aCγ1-containing molecule (e.g., an IgG1 fusion protein). In anotherembodiment, a mutated IgC region (of any isotype) is used in the fusionprotein and the modified effector function can be assessed relative toan antibody or Ig fusion protein containing the non-mutated form of theIgC region.

The CTLA4-immunoglobulin fusion proteins of the invention are useful forinhibiting the interaction of CTLA4 ligands (e.g., B7 family memberssuch as B7-1 and B7-2) with receptors on T cells (e.g., CD28 and/orCTLA4) to thereby inhibit delivery of a costimulatory signal in the Tcells and thus downmodulate an immune response. Use of theCTLA4-immunoglobulin fusion proteins of the invention is applicable in avariety of situations, such as to inhibit transplant rejection orautoimmune reactions in a subject. In these situations, immunoglobulinconstant region-mediated biological effector mechanisms, such ascomplement-mediated cell lysis. Fc receptor-mediated phagocytosis orantibody-dependent cellular cytotoxicity, may induce detrimental sideeffects in the subject and are therefore undesirable. TheCTLA4-immunoglobulin fusion proteins of the invention exhibit reducedIgC region-mediated effector functions compared to aCTLA4-immunoglobulin fusion protein in which the IgG1 constant region isused and, thus are likely to have improved immunoinhibitory properties.These compositions can also be used for immunomodulation, to produceanti-CTLA4 antibodies, to purify CTLA4 ligands and in screening assays.The CTLA4-Ab fusion proteins are particularly useful when bivalentpreparations are preferred, i.e. when crosslinking is desired.

One aspect of the invention pertains to isolated nucleic acid moleculesencoding modified CTLA4-immunoglobulin fusion proteins. The nucleicacids of the invention comprise a nucleotide sequence encoding a firstpeptide having a CTLA4 activity and a nucleotide sequence encoding asecond peptide comprising an immunoglobulin constant region which ismodified to reduce at least one constant region-mediated biologicaleffector function. A peptide having a CTLA4 activity is defined hereinas a peptide having at least one biological activity of the CTLA4protein, e.g., the ability to bind to the natural ligand(s) of the CTLA4antigen on immune cells, such as B7-1 and/or B7-2 on B cells, or otherknown or as yet undefined ligands on immune cells, and inhibit (e.g.,block) or interfere with immune cell mediated responses. In oneembodiment, the peptide having a CTLA4 activity binds B7-1 and/or B-2and comprises an extracellular domain of the CTLA4 protein. Preferably,the extracellular domain includes amino acid residues 20-144 of thehuman CTLA4 protein (amino acid positions 20-144 of SEQ ID NO: 24, 26and 28).

The present invention also contemplates forms of the extracellulardomain of CTLA4 which are expressed without Ig constant regions and areexpressed in E. coli. These soluble forms of the CTLA4 extracellulardomain, although not glycosylated, are fully functional and have similaruses as the CTLA4 immunoglobulin fusion proteins of the invention.

The nucleic acids of the invention further comprise a nucleotidesequence encoding a second peptide comprising an immunoglobulin constantregion which is modified to reduce at least one Ig constantregion-mediated biological effector function. Preferably, theimmunoglobulin constant region comprises a hinge region, a CH2 domainand a CH3 domain derived from Cγ1, Cγ2, Cγ3 or Cγ4. In one embodiment,the constant region segment is altered (e.g., mutated at specific aminoacid residues by substitution, deletion or addition of amino acidresidues) to reduce at least one IgC region-mediated effector function.In another embodiment, a constant region other than Cγ1 that exhibitsreduced IgC region-mediated effector functions relative to Cγ1 is usedin the fusion protein. In a preferred embodiment, the CH2 domain ismodified to reduce a biological effector function, such as complementactivation, Fc receptor interaction, or both complement activation andFc receptor interaction. For example, to reduce Fc receptor interaction,at least one amino acid residue selected from a hinge link region of theCH2 domain (e.g., amino acid residues at positions 234-237 of an intactheavy chain protein) is modified by substitution, addition or deletionof amino acids. In another embodiment, to reduce complement activationability, a constant region which lacks the ability to activatecomplement, such as Cγ4 or Cγ2 is used in the fusion protein (instead ofa Cγ1 constant region which is capable of activating complement). Inanother embodiment the variable region of the heavy and light chain isreplaced with a polypeptide having CTLA4 activity creating a CTLA4-Abmolecule. In a preferred embodiment the heavy chain constant region ofthe CTLA4-Ab molecule comprises a hinge region, a CH2 domain and a CH3domain derived from Cγ1, Cγ2, Cγ3 or Cγ4. In a preferred embodiment thelight chain constant region of the CTLA4-Ab molecule comprises an Igsignal sequence, the CTLA4 extracellular domain, and the light chain(kappa or lambda) constant domain.

The nucleic acids obtained in accordance with this invention can beinserted into various expression vectors, which in turn direct thesynthesis of the corresponding protein in a variety of hosts,particularly eucaryotic cells, such as mammalian or insect cell cultureand procaryotic cells, such as E. coli. Expression vectors within thescope of the invention comprise a nucleic acid as described herein and apromotor operably linked to the nucleic acid. Such expression vectorscan be used to transfect host cells to thereby produce fusion proteinsencoded by nucleic acids as described herein.

Another aspect of the invention pertains to isolatedCTLA4-immunoglobulin fusion proteins comprising a first peptide having aCTLA4 activity and a second peptide comprising an immunoglobulinconstant region which is modified to reduce at least one constantregion-mediated biological effector function relative to a CTLA4-IgG1fusion protein. A preferred CTLA4-immunoglobulin fusion proteincomprises an extracellular domain of the CTLA4 protein (e.g., amino acidpositions 20-144 of the human CTLA4-immunoglobulin fusion protein shownin SEQ ID NO: 24, 26 and 28) linked to an immunoglobulin constant regioncomprising a hinge region, a CH2 domain and a CH3 domain derived fromCγ1, Cγ2, Cγ3 or Cγ4. A preferred constant domain used to reduce thecomplement activating ability of the fusion protein is Cγ4. In oneembodiment, the CH2 domain of the immunoglobulin constant region ismodified to reduce at least one biological effector function, such ascomplement activation or Fc receptor interaction. In a particularlypreferred embodiment, the CTLA4-immunoglobulin fusion protein includes aCH2 domain which is modified by substitution of an amino acid residue atposition 234, 235 and/or 237 of an intact heavy chain protein. Oneexample of such a protein is a CTLA4-immunoglobulin fusion protein fusedto IgG4 comprising an amino acid sequence shown in SEQ ID NO: 28 or aCTLA4-immunoglobulin fused to IgG1 fusion protein comprising an aminoacid sequence shown in SEQ ID NO: 24.

The CTLA4-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a CTLA4 ligand (e.g., B7-1and/or B7-2) and a receptor therefor (e.g., CD28 and/or CTLA4) on thesurface of a T cell, to thereby suppress cell-mediated immune responsesin vivo. Inhibition of the CTLA4 ligand/receptor interaction may beuseful for both general immunosuppression and to induce antigen-specificT cell tolerance in a subject for use in preventing transplantationrejection (solid organ, skin and bone marrow) or graft versus hostdisease, particularly in allogeneic bone marrow transplantation. TheCTLA4-immunoglobulin fusion proteins can also be used therapeutically inthe treatment of autoimmune diseases, allergy and allergic reactions,transplantation rejection and established graft versus host disease in asubject. Moreover, the CTLA4-immunoglobulin fusion proteins of theinvention can be used as immunogens to produce anti-CTLA4 antibodies ina subject, to purify CTLA4 ligands and in screening assays to identifymolecules which inhibit the interaction of CTLA4 with a CTLA4 ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the “zip up” polymerase chain reaction(PCR) procedure used to contrust gene fusions.

FIG. 2A-B show the amino acid mutations introduced into the hinge andCH2 domains of hCTLA4-IgG1m (panel A) and hCTLA4-IgG4m (panel B).Mutated amino acid residues are underlined.

FIG. 3 is a schematic diagram of the expression vector pNRDSH.

FIG. 4A-B are graphic representations of competition ELISAs depictingthe ability of unlabeled hCTLA4-IgG1 or unlabeled hCTLA4-IgG4m tocompete for the binding of biotinylated hCTLA4-IgG1 to hB7-1-Ig (panelA) or hB7-2-Ig (panel B).

FIG. 5A-B are graphic representations of Fc receptor binding assaysdepicting the ability of CTLA4-IgG1 or CTLA4-IgG4 to bind to Fcreceptors. In panel A, the ability of unlabeled CTLA4-IgG1 or unlabeledCTLA4-IgG4 to compete for the binding of ¹²⁵I-labeled CTLA4-IgG1 toFcRI-positive U937 cells is depicted. In panel B, the percent specificactivity of unlabeled CTLA4-IgG1, CTLA4-IgG4 or hIgG1 used to competeitself for binding to U937 cells is depicted.

FIG. 6A-C are graphic representations of complement activation assaysdepicting the ability of CTLA4-IgG1, CTLA4-IgG4m or anti-B7-1 mAb (4B2)to activate complement-mediated lysis of CHO-B7-1 cells. In panel A,guinea pig complement is used as the complement source. In panel B,human serum is used as the complement source. In panel C, controluntransfected CHO cells are used as the target for complement-mediatedlysis.

FIG. 7 is a graphic representation of the binding of CTLA4-IgG1,CTLA4-IgG4m or anti-B7-1 mAb (4B2) to CHO-B7-1 cells, demonstrating thatdespite the inability of CTLA4-IgG4m to activate complement it can stillbind to CHO-B7-1 cells.

FIG. 8 is a graphic representation of a competition curve demonstratingthat soluble CTLA4 expressed in E. coli is functional and competes withunlabeled CTLA4Ig for binding to plate-bound B7-1.

DETAILED DESCRIPTION OF THE INVENTION

This invention features isolated CTLA4-immunoglobulin fusion proteinswhich have been modified to reduce immunoglobulin constant (IgC)region-mediated effector functions. The invention also features isolatednucleic acids encoding the proteins, methods for producing theCTLA4-immunoglobulin fusion proteins of the invention and methods forusing the CTLA4-immunoglobulin fusion proteins of the invention forimmunomodulation. E. coli-expressed forms of CTLA4 are also disclosed.These and other aspects of the invention are described in further detailin the following subsections:

I. Chimeric CTLA4-Immunoglobulin Gene Fusions

The invention provides isolated nucleic acids encodingCTLA4-immunoglobulin fusion proteins. The CTLA4-immunoglobulin fusionproteins are comprised of two components: a first peptide having a CTLA4activity and a second peptide comprising an immunoglobulin constantregion which, in certain embodiments is modified to reduce at least oneconstant region-mediated biological effector function. Accordingly, theisolated nucleic acids of the invention comprise a first nucleotidesequence encoding the first peptide having a CTLA4 activity and a secondnucleotide sequence encoding the second peptide comprising animmunoglobulin constant region which, in a preferred embodiment, ismodified to reduce at least one constant region-mediated biologicaleffector function. In the case of CTLA4Ig forms, the first and secondnucleotide sequences are linked (i.e., in a 5′ to 3′ orientation byphosphodiester bonds) such that the translational frame of the CTLA4 andIgC coding segments are maintained (i.e., the nucleotide sequences arejoined together in-frame). Thus, expression (i.e., transcription andtranslation) of the nucleotide sequences produces a functional CTLA4Igfusion protein. In the case of the CTLA4-Ab fusion proteins, the heavychain gene is constructed such that the CTLA4 extracellular bindingdomain is linked to a 5′ signal sequence and a 3′ immunoglobulin CH1,hinge, CH2, and CH3 domain. CTLA4-light chain constructs are prepared inwhich an Ig signal sequence, an intron, the CTLA4 extracellular domain,an intron, and the light chain constant domain are linked. The DNAencoding the heavy and light chains is then expressed using anappropriate expression vector as described in the Examples.

The term “nucleic acid” as used herein is intended to include fragmentsor equivalents thereof. The term “equivalent” is intended to includenucleotide sequences encoding functionally equivalentCTLA4-immunoglobulin fusion proteins, i.e., proteins which have theability to bind to the natural ligand(s) of the CTLA4 antigen on immunecells, such as B7-1 and/or B7-2 on B cells, and inhibit (e.g., block) orinterfere with immune cell mediated responses.

The term “isolated” as used throughout this application refers to anucleic acid or fusion protein substantially free of cellular materialor culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Anisolated nucleic acid is also free of sequences which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the organism from which the nucleic acid is derived.

The nucleic acids of the invention can be prepared by standardrecombinant DNA techniques. For example, a chimeric CTLA4-immunoglobulingene fusion can be constructed using separate template DNAs encodingCTLA4 and an immunoglobulin constant region and a “zip up” polymerasechain reaction (PCR) procedure as described in Example 1 and illustratedschematically in FIG. 1. Alternatively, a nucleic acid segment encodingCTLA4 can be ligated in-frame to a nucleic acid segment encoding animmunoglobulin constant region using standard techniques. A nucleic acidof the invention can also be chemically synthesized using standardtechniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis whichhas been automated in commercially available DNA synthesizers (See e.g.,Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No.4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,incorporated by reference herein).

The nucleic acid segments of the CTLA4-immunoglobulin gene fusions ofthe invention are described in further detail below:

A. CTLA4 Gene Segment

An isolated nucleic acid of the invention encodes a first peptide havinga CTLA4 activity. The phrase “peptide having a CTLA4 activity” or“peptide having an activity of CTLA4” is used herein to refer to apeptide having at least one biological activity of the CTLA4 protein,i.e., the ability to bind to the natural ligand(s) of the CTLA4 antigenon immune cells, such as B7-1 and/or B7-2 on B cells, or other known oras yet undefined ligands on immune cells, and which, in soluble form,can inhibit (e.g., block) or interfere with immune cell mediatedresponses. In one embodiment, the CTLA4 protein is a human CTLA4protein, the nucleotide and amino acid sequences of which are disclosedin Harper, K., et al. (1991) J. Immunol. 147:1037-1044 and Dariavich, etal. (1988) Eur. J. Immunol. 18(12):1901-1905. In another embodiment, thepeptide having a CTLA4 activity binds B7-1 and/or B7-2 and comprises atleast a portion of an extracellular domain of the CTLA4 protein.Preferably, the extracellular domain includes amino acid residues 1-125of the human CTLA4 protein (amino acid positions 20-144 of SEQ ID NO:24, 26 and 28). CTLA4 proteins from other species (e.g., mouse) are alsoencompassed by the invention. The nucleotide and amino acid sequences ofmouse CTLA4 are disclosed in Brunet, J. F., et al., (1987) Nature328:267-270.

The nucleic acids of the invention can be DNA or RNA. Nucleic acidencoding a peptide having a CTLA4 activity may be obtained from mRNApresent in activated T lymphocytes. It is also possible to obtainnucleic acid encoding CTLA4 from T cell genomic DNA. For example, thegene encoding CTLA4 can be cloned from either a cDNA or a genomiclibrary in accordance with standard protocols. A cDNA encoding CTLA4 canbe obtained by isolating total mRNA from an appropriate cell line.Double stranded cDNAs can then prepared from the total mRNA.Subsequently, the cDNAs can be inserted into a suitable plasmid orbacteriophage vector using any one of a number of known techniques.Genes encoding CTLA4 can also be cloned using established polymerasechain reaction techniques in accordance with the nucleotide sequenceinformation provided by the invention (see Example 1). For example, aDNA vector containing a CTLA4 cDNA can be used as a template in PCRreactions using oligonucleotide primers designed to amplify a desiredregion of the CTLA4 cDNA, e.g., the extracellular domain, to obtain anisolated DNA fragment encompassing this region using standardtechniques.

It will be appreciated by those skilled in the art that variousmodifications and equivalents of the nucleic acids encoding theCTLA4-immunoglobulin fusion proteins of the invention exist. Forexample, different cell lines can be expected to yield DNA moleculeshaving different sequences of bases. Additionally, variations may existdue to genetic polymorphisms or cell-mediated modifications of thegenetic material. Furthermore, the nucleotide sequence of aCTLA4-immunoglobulin fusion protein of the invention can be modified bygenetic techniques to produce proteins with altered amino acid sequencesthat retain the functional properties of CTLA4 (e.g., the ability tobind to B7-1 and/or B7-2). Such sequences are considered within thescope of the invention, wherein the expressed protein is capable ofbinding a natural ligand of CTLA4 and, when in the appropriate form(e.g., soluble) can inhibit B7:CD28/CTLA4 interactions and modulateimmune responses and immune function. In addition, it will beappreciated by those of skill in the art that there are other B7-bindingligands and the fusion of these alternative molecules (such as CD28) toform immuonglobulin fusion proteins or expressed in soluble form in E.coli is also contemplated by the present invention.

To express a CTLA4-immunoglobulin fusion protein of the invention, thechimeric gene fusion encoding the CTLA4-immunoglobulin fusion proteintypically includes a nucleotide sequence encoding a signal sequencewhich, upon transcription and translation of the chimeric gene, directssecretion of the fusion protein. A native CTLA4 signal sequence (e.g.,the human CTLA4 signal sequence disclosed in Harper, K., et al. (1991)J. Immunol. 147.1037-1044) can be used or alternatively, a heterologoussignal sequence can be used. For example, the oncostatin-M signalsequence (Malik N., et al. (1989) Mol. Cell. Biol. 9(7), 2847-2853) oran immunoglobulin signal sequence (e.g., amino acid positions 1 to 19 ofSEQ ID NO: 24, 26 and 28) can be used to direct secretion of aCTLA4-immunoglobulin fusion protein of the invention. A nucleotidesequence encoding a signal sequence can be incorporated into thechimeric gene fusion by standard recombinant DNA techniques, such as by“zip up” PCR (described further in Example 1) or by ligating a nucleicacid fragment encoding the signal sequence in-frame at the 5′ end of anucleic acid fragment encoding CTLA4.

B. Immunoglobulin Gene Segment

The CTLA4-immunoglobulin fusion protein of the invention furthercomprises a second peptide linked to the peptide having a CTLA4activity. In one embodiment the second peptide comprises a light chainconstant region. In a preferred embodiment the light chain is a kappalight chain.

In another embodiment the second peptide comprises a heavy chainconstant region. In a preferred embodiment the constant region comprisesan immunoglobulin hinge region, a CH2 domain and a CH3 domain. Inanother embodiment the constant region also comprises a CH1 domain. Theconstant region is preferably derived from Cγ1, Cγ2, Cγ3 or Cγ4. In apreferred embodiment the heavy chain constant region is modified toreduce at least one constant region-mediated biological effectorfunction. In one embodiment, the constant region segment (either Cγ1 oranother isotype) is altered (e.g., mutated from the wild-type sequenceat specific amino acid residues by substitution, deletion or addition ofamino acid residues) to reduce at least one IgC region-mediated effectorfunction. The effector functions of this altered fusion protein can beassessed relative to an unaltered IgC region-containing molecule (e.g.,a whole antibody or Ig fusion protein). In another embodiment, aconstant region other than Cγ1 that exhibits reduced IgC region-mediatedeffector functions is used in the fusion protein. The effector functionsof this fusion protein can be assessed relative to a Cγ1-containingmolecule (e.g., an IgG1 antibody or IgG1 fusion protein). In aparticularly preferred embodiment, the fusion protein comprises aconstant region other than Cγ1 that is also mutated to further reduceeffector function. For example, a preferred IgC region is a mutated Cγ4region.

The term “immunoglobulin constant (IgC) region-mediated biologicaleffector function” is intended to include biological responses whichrequire or involve, at least in part, the constant region of animmunoglobulin molecule. Examples of such effector functions includecomplement activation. Fc receptor interactions, opsonization andphagocytosis, antibody-dependent cellular cytotoxicity (ADCC), releaseof reactive oxygen intermediates and placental transfer. While sucheffector functions are desirable in many immune responses, they areundesirable in situations where an immune response is to bedownmodulated. The CTLA4-immunoglobulin fusion proteins of the inventionexhibit reduced IgC region-mediated biological effector functions andthus are efficient agents for downregulating immune responses.Additionally, the CTLA4-immunoglobulin fusion proteins of the inventiondisplay a long plasma half life in vivo. The long plasma half-life makesthe proteins particularly useful as therapeutic agents.

All immunoglobulins have a common core structure of two identical lightand heavy chains held together by disulfide bonds. Both the light chainsand the heavy chains contain a series of repeating, homologous units,each about 110 amino acid residues in length, which fold independentlyin a common globular motif, called an immunoglobulin domain. In eachchain, one domain (V) has a variable amino acid sequence depending onthe antibody specificity of the molecule. The other domains (C) have aconstant sequence common among molecules of the same isotype. Heavychains are designated by the letter of the Greek alphabet correspondingto the overall isotype of the antibody: IgA1 contains α1 heavy chains:IgA2, α2; IgD, δ; IgE, ε; IgG1, γ1; IgG2, γ2; IgG3, γ3; IgG4, γ4; andIgM, μ. Each heavy chain includes four domains; an amino terminalvariable, or VH domain which displays the greatest sequence variationamong heavy chains and three domains which form the constant region(CH1, CH2 and CH3) in order from the amino to the carboxy terminus ofthe heavy chain. In γ, α and δ heavy chains, there is a nonglobularregion of amino acid sequence, known as the hinge, located between thefirst and second constant region domains (CH1 and CH2) permitting motionbetween these two domains.

To modify a CTLA4-immunoglobulin fusion protein such that it exhibitsreduced binding to the FcRI receptor, the immunoglobulin constant regionsegment of the CTLA4-immunoglobulin fusion protein can be mutated atparticular regions necessary for Fc receptor (FcR) interactions (seeCanfield, S. M. and S. L. Morrison (1991) J. Exp. Med. 173:1483-1491;and Lund, J. et al. (1991) J. of Immunol. 147:2657-2662). Reduction inFcR binding ability of a CTLA4-immunoglobulin fusion protein will alsoreduce other effector functions which rely on FcR interactions, such asopsonization and phagocytosis and antigen-dependent cellularcytotoxicity. To reduce FcR binding, in one embodiment, the constantregion is mutated within a region of the CH2 domain referred to as the“hinge link” or “lower hinge” region. This region encompasses amino acidresidues 234-239 in a full-length native immunoglobulin heavy chain. Itshould be appreciated that all IgC region amino acid residue positionsdescribed herein refer to the position within the full-length intactnative immunoglobulin heavy chain; it will be apparent to those skilledin the art that depending upon the length of the CTLA4 segment used inthe CTLA4-immunoglobulin fusion protein, the positions of thecorresponding IgC amino acid residues within the fusion protein willvary (Kabat, E. A. T. T. Wu, M. Reid-Miller, H. M. Perry, and K. S.Gottesman eds. (1987) “Sequences of Proteins of Immunological Interest”National Institutes of Health, Bethesda, Md.). The hinge link region canbe mutated by substitution, addition or deletion of amino acid residues.A preferred CTLA4-immunoglobulin fusion protein of the invention is onein which the IgG1 constant region has substitution mutations atpositions 234, 235 and/or 237 of the Cγ1 segment. Preferably, Leu at 234is substituted with Ala, Leu at 235 is substituted with Glu and Gly at237 is substituted with Ala (see Example 1). A preferredCTLA4-immunoglobulin fusion protein of the invention is one in whichIgG4 has substitution mutations at positions 235 and/or 237 of the Cγ4segment. Preferably, Leu at 235 is substituted with Glu and Gly at 237is substituted with Ala (see Example 1).

In another embodiment, the Fc receptor binding capability of theCTLA4-immunoglobulin fusion protein is reduced by mutating a region ofthe CH2 domain referred to as the “hinge-proximal bend” region (aminoacid residues at positions 328-333 within a full-length intact heavychain). This region can be mutated by substitution, addition or deletionof amino acid residues. In a preferred embodiment, position 331 of Cγ1or Cγ3 is mutated. A preferred mutation in Cγ1 or Cγ3 is substitution ofPro with Ser.

To modify a CTLA4-immunoglobulin fusion protein such that it exhibitsreduced complement activation ability, the immunoglobulin constantregion segment of the fusion protein can be mutated at particularregions important for complement activation, such as regions involved inIgC region binding to the C1q component of complement. In oneembodiment, one or more residues present within the CH2 domain of IgGsubclasses that are involved in C1q binding are altered. In a preferredembodiment, positions 318, 320 and/or 322 are mutated (see Duncan andWinter (1988) Nature 332, 738-740). Preferably, Glu at 318 issubstituted with Ala or Val, Lys at 320 is substituted with Ala or Glnand/or Lys at 322 is substituted with Ala or Gln.

Alternatively, to reduce complement activation by theCTLA4-immunoglobulin fusion protein, a constant region which lacks theability to activate complement can be used in the fusion protein. Forexample, it is known that both IgG1 and IgG3, but not IgG2 and IgG4activate the classical complement cascade in the presence of humancomplement. Accordingly, a CTLA4-immunoglobulin fusion protein utilizinga Cγ4 constant region exhibits reduced complement activation abilityrelative to a CTLA4-immunoglobulin fusion protein comprising IgG1 (asdemonstrated in Example 2).

In yet another embodiment, the hinge region of the IgC segment isaltered to inhibit complement activation ability. The hinge regions ofthe human IgG molecules vary in amino acid sequence and composition aswell as length. For example, IgG1, IgG2 and IgG4 have hinge regionsconsisting of 12 to 15 amino acids, whereas IgG3 has an extended hingeregion, consisting of 62 amino acids. The hinge region is believed to beessential for binding with the first component of complement, C1q (seeTan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162-166). A number ofchimeric human IgG3 and IgG4 molecules with different hinge lengths andamino acid composition have been produced, confirming the role of thehinge region in C1q binding and complement activation. To reduce orinterfere with the ability of a CTLA4-immunoglobulin (IgG1) orCTLA4-immunoglobulin (IgG3) construct to activate complement, it may benecessary to modify, by substitution, addition or deletion, at least oneamino acid residue in the hinge region. In one embodiment, the hingeregion of Cγ1 or Cγ3 is substituted with a hinge region derived from Cγ2or Cγ4, each of which lack the ability to activate complement.

In addition to modifying the CTLA4-immunoglobulin fusion proteins of theinvention to reduce IgC region-mediated biological effector functions,the fusion proteins can be further modified for other purposes, e.g., toincrease solubility, enhance therapeutic or prophylactic efficacy, orstability (e.g., shelf life ex vivo and resistance to proteolyticdegradation in vivo). Such modified proteins are considered functionalequivalents of the CTLA4-immunoglobulin fusion proteins as definedherein. For example, amino acid residues of the CTLA4 portion of thefusion protein which are not essential for CTLA4 ligand interaction canbe modified by being replaced by another amino acid whose incorporationmay enhance, diminish, or not affect reactivity of the fusion protein.Alternatively, a CTLA4-immunoglobulin fusion protein which binds onlyB7-1 or B7-2 but not both could be created by mutating residues involvedin binding to one ligand or the other. Another example of a modificationof a CTLA4-immunoglobulin fusion protein is substitution of cysteineresidues, preferably with alanine, serine, threonine, leucine orglutamic acid residues, to minimize dimerization via disulfide linkages.A particularly preferred modification is substitution of cysteineresidues in the hinge region of the immunoglobulin constant region withserine. In addition, amino acid side chains of a CTLA4-immunoglobulinfusion protein can be chemically modified.

A particularly preferred embodiment of the invention features a nucleicacid encoding a CTLA4-immunoglobulin fusion protein comprising anucleotide sequence encoding a first peptide having a CTLA4 activity anda nucleotide sequence encoding a second peptide comprising an IgG4immunoglobulin constant region, Cγ4. Preferably, the nucleic acid is aDNA and the first peptide comprises an extracellular region of CTLA4which binds B7-1. Such a CTLA4-IgG4 construct can comprise a nucleotidesequence show in SEQ ID NO: 25 and an amino acid sequence shown in SEQID NO: 26. In an even more preferred embodiment, the CH2 domain of theCγ4 portion of this CTLA4IgG4 fusion protein is modified to reduce Fcreceptor interaction. For example, the CH2 domain can be modified bysubstitution of Leu at position 235 (e.g., with Glu) and/or substitutionof Gly at position 237 (e.g., with Ala). A particularly preferredCTLA4-IgG4 fusion protein comprises the extracellular domain of humanCTLA4 (i.e., amino acid residues 1-125), has reduced Fc receptorinteraction due to two substitutions in the CH2 domain (i.e.,substitution of Leu at position 235 with Glu and substitution of Gly atposition 237 with Ala). Such a CTLA4-IgG4 fusion protein comprises anamino acid sequence shown in SEQ ID NO: 28 and a nucleotide sequenceshown in SEQ ID NO: 27. This construct, referred to as CTLA4-IgG4m,exhibits markedly reduced complement activation ability and FcR bindingactivity relative to a wild-type CTLA4-IgG1 construct (see Example 2).

Another preferred embodiment of the invention features a nucleic acidencoding a CTLA4-IgG1 fusion protein comprising a nucleotide sequenceencoding a first peptide having a CTLA4 activity and a nucleotidesequence encoding a second peptide comprising an immunoglobulin constantregion, Cγ1, which is modified to reduce at least one constantregion-mediated biological effector functions. Preferably, the nucleicacid is a DNA and the first peptide comprises an extracellular region ofCTLA4 which binds B7-1. To reduce Fc receptor interaction the CH2 domainof Cγ1 is modified by substitution of one or more of the following aminoacid residues: Leu at position 235; Leu at position 234; and Gly atposition 237. A particularly preferred CTLA4-IgG1 fusion proteincomprises the extracellular domain of human CTLA4 (i.e., amino acidresidues 1-125), has reduced Fc receptor interaction due to threesubstitutions in the CH2 domain (i.e., substitution of Leu at position234 with Ala, substitution of Leu at position 235 with Glu andsubstitution of Gly at position 237 with Ala). Such a CTLA4-IgG1 fusionprotein, referred to herein as CTLA4-IgG1m, comprises an amino acidsequence shown in SEQ ID NO: 24 and a nucleotide sequence shown in SEQID NO: 23.

Nucleic acid encoding a peptide comprising an immunoglobulin constantregion can be obtained from human immunoglobulin mRNA present in Blymphocytes. It is also possible to obtain nucleic acid encoding animmunoglobulin constant region from B cell genomic DNA. For example, DNAencoding Cγ1 or Cγ4 can be cloned from either a cDNA or a genomiclibrary or by polymerase chain reaction (PCR) amplification inaccordance with protocols herein described. The nucleic acids of theinvention can be DNA or RNA. A preferred nucleic acid encoding animmunoglobulin constant region comprises all or a portion of thefollowing: the DNA encoding human Cγ1 (Takahashi, N. S. et al. (1982)Cell 29:671-679), the DNA encoding human Cγ2 (Kabat, E. A. T. T. Wu, M.Reid-Miller, H. M. Perry, and K. S. Gottesman eds. (1987) “Sequences ofProteins of Immunological Interest” National Institutes of Health,Bethesda, Md.); the DNA encoding human Cγ3 (Huck, S., et al. (1986)Nucl. Acid Res. 14:1779); and the DNA encoding human Cγ4 (Kabat et al.,supra).

A number of processes are known in the art for modifying a nucleotide oramino acid sequence to thereby mutate the IgC regions as describedherein. For example, mutations can be introduced into a DNA by any oneof a number of methods, including those for producing simple deletionsor insertions, systematic deletions, insertions or substitutions ofclusters of bases or substitutions of single bases, to generateCTLA4-immunoglobulin fusion proteins of the invention and equivalentsthereof. Preferably, amino acid substitutions, deletions or additions,such as in the CH2 domain of the immunoglobulin constant region, arecreated by PCR mutagenesis as described in Example 1 or by standardsite-directed mutagenesis. Site directed mutagenesis systems are wellknown in the art. For example, protocols and reagents can be obtainedcommercially from Amersham International PLC, Amersham, U.K.

II. Expression Vectors and Host Cells

The CTLA4-immunoglobulin fusion proteins of the invention can beexpressed by incorporating a chimeric CTLA4-immunoglobulin fusion genedescribed herein into an expression vector and introducing theexpression vector into an appropriate host cell. Accordingly, theinvention further pertains to expression vectors containing a nucleicacid encoding a CTLA4-immunoglobulin fusion protein and to host cellsinto which such expression vectors have been introduced. An expressionvector of the invention, as described herein, typically includesnucleotide sequences encoding the CTLA4-immunoglobulin fusion proteinoperably linked to at least one regulatory sequence. “Operably linked”is intended to mean that the nucleotide sequence is linked to aregulatory sequence in a manner which allows expression of thenucleotide sequence in a host cell (or by a cell extract). Regulatorysequences are art-recognized and can be selected to direct expression ofthe desired protein in an appropriate host cell. The term regulatorysequence is intended to include promoters, enhancers, polyadenylationsignals and other expression control elements. Such regulatory sequencesare known to those skilled in the art and are described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transfected and/or the type and/or amount of protein desiredto be expressed.

An expression vector of the invention can be used to transfect cells,either procaryotic or eucaryotic (e.g., mammalian, insect or yeastcells) to thereby produce fusion proteins encoded by nucleotidesequences of the vector. Expression in procaryotes is most often carriedout in E. coli with vectors containing constitutive or induciblepromoters. Certain E. coli expression vectors (so called fusion-vectors)are designed to add a number of amino acid residues to the expressedrecombinant protein, usually to the amino terminus of the expressedprotein. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the target recombinant protein; and 3) to aid in thepurification of the target recombinant protein by acting as a ligand inaffinity purification. Examples of fusion expression vectors includepGEX (Amrad Corp., Melbourne, Australia) and pMAL (New England Biolabs,Beverly, Mass.) which fuse glutathione S-tranferase and maltose Ebinding protein, respectively, to the target recombinant protein.Accordingly, a chimeric CTLA4-immunoglobulin fusion gene may be linkedto additional coding sequences in a procaryotic fusion vector to aid inthe expression, solubility or purification of the fusion protein. Often,in fusion expression vectors, a proteolytic cleavage site is introducedat the junction of the fusion moiety and the target recombinant proteinto enable separation of the target recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69:301-315) and pET 11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). Target gene expression from the pTrc vector relies onhost RNA polymerase transcription from the hybrid trp-lac fusionpromoter. Target gene expression from the pET 11d vector relies ontranscription from the T-7 gn10-lac 0 fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize expression of recombinant CTLA4-immunoglobulinfusion protein in E. coli is to express the protein in a host bacteriawith an impaired capacity to proteolytically cleave the recombinantprotein (Gottesman, S., Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).Another strategy would be to alter the nucleotide sequence of theCTLA4-immunoglobulin fusion protein to be inserted into an expressionvector so that the individual codons for each amino acid would be thosepreferentially utilized in highly expressed E. coli proteins (Wada etal., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences are encompassed by the invention and can be carried outby standard DNA synthesis techniques.

In another preferred embodiment a soluble CTLA4 extracellular domain isexpressed in E coli using an appropriate expression vector. These forms,although not glycosylated, remain fully functional and represent anadvantage because of the ease with which bacterial cells are grown.

Alternatively, a CTLA4-immunoglobulin fusion protein can be expressed ina eucaryotic host cell, such as mammalian cells (e.g., Chinese hamsterovary cells (CHO) or NS0 cells), insect cells (e.g., using a baculovirusvector) or yeast cells. Other suitable host cells may be found inGoeddel, (1990) supra or are known to those skilled in the art.Eucaryotic, rather than procaryotic, expression of aCTLA4-immunoglobulin fusion protein may be preferable since expressionof eucaryotic proteins in eucaryotic cells can lead to partial orcomplete glycosylation and/or formation of relevant inter- orintra-chain disulfide bonds of a recombinant protein. For expression inmammalian cells, the expression vector's control functions are oftenprovided by viral material. For example, commonly used promoters arederived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.To express a CTLA4-immunoglobulin fusion protein in mammalian cells,generally COS cells (Gluzman, Y., (1981) Cell 23:175-182) are used inconjunction with such vectors as pCDM8 (Seed, B., (1987) Nature 329:840)for transient amplification/expression, while CHO (dhfr⁻ Chinese HamsterOvary) cells are used with vectors such as pMT2PC (Kaufman et al.(1987), EMBO J. 6:187-195) for stable amplification/expression inmammalian cells. A preferred cell line for production of recombinantprotein is the NSO myeloma cell line available from the ECACC (catalog#85110503) and described in Galfre, G. and Milstein, C. ((1981) Methodsin Enzymology 73(13):3-46; and Preparation of Monoclonal Antibodies:Strategies and Procedures, Academic Press, New York, N.Y.). Examples ofvectors suitable for expression of recombinant proteins in yeast (e.g.,S. cerivisae) include pYepSec1 (Baldari, et al., (1987) Embo J.6:229-234). pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943). pJRY88(Schultz et al., (1987) Gene 54:113-123), and pYES2 (InvitrogenCorporation, San Diego, Calif.). Baculovirus vectors available forexpression of proteins in cultured insect cells (SF 9 cells) include thepAc series (Smith et al., (1983) Mol. Cell Biol. 3:2156-2165) and thepVL series (Lucklow, V. A., and Summers, M. D., (1989) Virology170:31-39).

Vector DNA can be introduced into procaryotic or eucaryotic cells viaconventional transformation or transfection techniques such as calciumphosphate or calcium choloride co-precipitation. DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory textbooks.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfaction of cells may integrate DNA into their genomes. In order toidentify and select these integrants, a gene that encodes a selectablemarker (e.g., resistance to antibiotics) is generally introduced intothe host cells along with the gene of interest. Preferred selectablemarkers include those which confer resistance to drugs, such as G418,hygromycin and methotrexate. Nucleic acid encoding a selectable markermay be introduced into a host cell on the same plasmid as the gene ofinterest or may be introduced on a separate plasmid. Cells containingthe gene of interest can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die). The surviving cells can then be screened forproduction of CTLA4-immunoglobulin fusion proteins by, for example,immunoprecipitation from cell supernatant with an anti-CTLA4 monoclonalantibody.

The invention also features methods of producing CTLA4-immunoglobulinfusion proteins. For example, a host cell transfected with a nucleicacid vector directing expression of a nucleotide sequence encoding aCTLA4-immunoglobulin fusion protein can be cultured in a medium underappropriate conditions to allow expression of the protein to occur. Inone embodiment, a recombinant expression vector containing DNA encodinga CTLA4-IgG1 fusion protein having modified constant region-mediatedeffector functions is produced. In another embodiment, a recombinantexpression vector containing DNA encoding a CTLA4-IgG4 fusion proteinhaving modified constant region-mediated effector functions is produced.In addition, one or more expression vectors containing DNA encoding, forexample, a CTLA4-IgG1 fusion protein and another fusion protein such asa CTLA4-IgG4 fusion protein can be used to transfect a host cell tocoexpress these fusion proteins. Fusion proteins produced by recombinanttechnique may be secreted and isolated from a mixture of cells andmedium containing the protein. Alternatively, the protein may beretained cytoplasmically and the cells harvested, lysed and the proteinisolated. A cell culture typically includes host cells, media and otherbyproducts. Suitable mediums for cell culture are well known in the art.Protein can be isolated from cell culture medium, host cells, or bothusing techniques known in the art for purifying proteins.

III. Isolation and Characterization of CTLA4-Immunoglobulin FusionProteins

Another aspect of the invention pertains to CTLA4-immunoglobulin fusionproteins having modified effector functions compared to a CTLA4-IgG1protein. Such proteins comprise a first peptide having a CTLA4 activityand a second peptide comprising an immunoglobulin constant region whichis modified to reduce at least one constant region-mediated biologicaleffector function relative to a CTLA4-IgG1 fusion protein. A peptidehaving a CTLA4 activity has been previously defined herein. In apreferred embodiment, the first peptide comprises an extracellulardomain of the human CTLA4 protein (e.g., amino acid residues 20-144 ofSEQ ID NO: 24, 26 and 28) and binds B7-1 and/or B7-2. The second peptidecomprising an immunoglobulin constant region preferably includes a hingeregion, a CH2 domain and a CH3 domain derived from Cγ1, Cγ2, Cγ3, orCγ4. Typically, the CH2 domain is modified to reduce constantregion-mediated biological effector functions, such as complementactivation and/or Fc receptor interaction as previously described indetail herein.

Another embodiment of the invention provides a substantially purepreparation of a CTLA4-immunoglobulin fusion protein as describedherein. Such a preparation is substantially free of proteins andpeptides with which the protein naturally occurs in a cell or with whichit naturally occurs when secreted by a cell.

CTLA4-immunoglobulin fusion proteins, expressed in mammalian cells orelsewhere, can be purified according to standard procedures of the art,including ammonium sulfate precipitation, fractionation columnchromatography (e.g., ion exchange, gel filtration, electrophoresis,affinity chromatography, etc.) and ultimately, crystallization (seegenerally, “Enzyme Purification and Related Techniques”, Methods inEnzymology, 22:233-577 (1971)). Preferably, the CTLA4-immunoglobulinfusion proteins are purified using an immobilized protein A column(Repligen Corporation, Cambridge, Mass.). Once purified, partially or tohomogeneity, the recombinantly produced CTLA4-immunoglobulin fusionproteins or portions thereof can be utilized in compositions suitablefor pharmaceutical administration as described in detail herein.

In one embodiment the CTLA4-immunoglobulin fusion protein is an antibodyform in which the heavy and light chains have been replaced with theextracellular domain of CTLA4. This molecule has a different valency andhigher affinity for CTLA4 ligands, thus making it possible to obtainsimilar results while using less of the agent. The fact that thismolecule has a true antibody tail which is fully glycosylated means thatnumerous cell lines for producing the CTLA4Ab fusion protein and dataregarding the clinical use of antibodies may be relied on.

Screening of CTLA4-immunoglobulin fusion proteins which have beenmodified to reduce at least one constant region-mediated biologicaleffector function as described herein can be accomplished using one ormore of several different assays which measure different effectorfunctions. For example, to identify a CTLA4-immunoglobulin fusionprotein having reduced Fc receptor interaction, a monomeric IgG bindingassay can be conducted (see Example 2). A cell which expresses an Fcreceptor, such as a mononuclear phagocyte or the U937 cell line (FcγRIexpression), a hematopoietic cell (FcγRII expression; Rosenfeld, S. I.,et al. (1985) J. Clin. Invest. 76:2317-2322) or a neutrophil (FcγRIIIexpression; Fleit, H. B., et al. (1982) Proc. Natl. Acad. Sci. USA79:3275-3279 and Petroni, K. C., et al. (1988) J. Immunol.140:3467-3472) is contacted with, for example, ¹²⁵I-labeledimmunoglobulin of the IgG1 isotype in the presence or absence of amodified CTLA4-immunoglobulin fusion protein of the invention and in thepresence or absence of an appropriate control molecule (e.g., anunlabeled IgG1 antibody or a CTLA4-IgG1 fusion protein). The amount of¹²⁵I-labeled IgG1 bound to the cells and/or the amount of free¹²⁵I-labeled IgG in the supernatant is determined. ACTLA4-immunoglobulin fusion protein having reduced Fc receptor bindingis identified by a reduced ability (or lack of ability) to inhibitbinding of the ¹²⁵I-labeled IgG1 to the cells (relative to the controlmolecule). Monomeric IgG binding assay are described further in Lund,J., et al. (1991) J. Immunol. 147:2657-2662; and Woof, J. M., et al.(1986) Mol. Immunol. 23:319.

To identify a CTLA4-immunoglobulin fusion protein with a reduced abilityto activate the complement cascade, a complement activation assay suchas that described in Example 2 can be used. In this assay, a cell whichexpresses a CTLA4 ligand (e.g., B7-1 or B7-2) on its surface is loadedwith a detectable substance, e.g., a fluorescent dye, and then contactedwith the CTLA4-immunoglobulin fusion protein and a complement source(e.g., purified guinea pig complement or human serum as a source ofhuman complement). Cell lysis, as determined by release of thefluorescent dye from the cells, is determined as an indication ofactivation of the complement cascade upon binding ofCTLA4-immunoglobulin to the CTLA4 ligand on the cell surface. Cellswhich do not express a CTLA4 ligand on their surface are used as anegative control. A CTLA4-immunoglobulin fusion protein with reducedability (or lack of the ability) to activate complement relative to anappropriate control molecule (e.g., anti-B7-1 antibody of the IgG1isotype or a CTLA4-IgG1 fusion protein) is identified by a reduction inor absence of cell lysis of labeled, CTLA4 ligand positive cells whenincubated in the presence of the CTLA4-immunoglobulin fusion protein ofthe invention and complement compared to cells incubated in the presenceof the control molecule and complement.

In another complement activation assay, the ability of aCTLA4-immunoglobulin fusion protein to bind the first component of thecomplement cascade, C1q, is assessed. For example, C1q binding can bedetermined using a solid phase assay in which ¹²⁵I-labeled human C1q isadded to an amount of CTLA4-immunoglobulin fusion protein complexed witha CTLA4 ligand, such as B7-1 or B7-2, and the amount of bound¹²⁵I-labeled human C1q quantitated. A CTLA4-immunoglobulin fusionprotein having a reduced complement activation activity (or lack ofcomplement activation activity) is identified by a reduction in orabsence of the ability to bind the ¹²⁵I-labeled human C1q relative to anappropriate control molecule (e.g., an IgG1 antibody or a CTLA4-IgG1fusion protein). C1q binding assays are described further in Tan, L. K.,et al. (1990) Proc. Natl. Acad. Sci. USA 87:162-166; and Duncan, A. R.and G. Winter (1988) Nature 332:738-740.

Additional assays for other immunoglobulin constant region-mediatedeffector functions, such as opsonization and phagocytosis,antibody-dependent cellular cytotoxicity and release of reactive oxygenintermediates, have been described in the art and are known to theskilled artisan.

Screening for CTLA4-immunoglobulin fusion proteins which have a CTLA4activity as described herein can be accomplished using one or more ofseveral different assays. For example, the fusion proteins can bescreened for specific reactivity with an anti-CTLA4 antibody (e.g., amonoclonal or polyclonal anti-CTLA4 antibody) or with a soluble form ofa CTLA4 ligand, such as a B7-1 or B7-2 fusion protein (e.g., B7-1Ig orB7-2Ig). For example, appropriate cells, such as CHO or NS0 cells, canbe transfected with a DNA encoding a CTLA4-immunoglobulin fusion proteinand the cell supernatant analyzed for expression of the resulting fusionprotein using an anti-CTLA4 monoclonal antibody or B7-1Ig or B7-2Igfusion protein in a standard immunoprecipitation assay. Alternatively,the binding of a CTLA4-immunoglobulin fusion protein to a cell whichexpresses a CTLA4 ligand, such as a B7-1 or B7-2, on its surface can beassessed. For example, a cell expressing a CTLA4 ligand, such as a CHOcell transfected to express B7-1, is contacted with theCTLA4-immunoglobulin fusion protein and binding detected by indirectimmunostaining using, for example, a FITC-conjugated reagent (e.g., goatanti-mouse Ig serum for murine monoclonal antibodies or goat anti-humanIgCγ serum for fusion proteins) and fluorescence analyzed by FACS®analysis (Becton Dickinson & Co., Mountain View, Calif.).

Other suitable assays take advantage of the functional characteristicsof the CTLA4-immunoglobulin fusion protein. As previously set forth, theability of T cells to synthesize cytokines depends not only on occupancyor cross-linking of the T cell receptor for antigen (“the primaryactivation signal provided by, for example antigen bound to an MHCmolecule, anti-CD3, or phorbol ester to produce an “activated T cell”),but also on the induction of a costimulatory signal, in this case, byinteraction of a B7 family protein (e.g., B7-1 or B7-2) with its ligand(CD28 and/or CTLA4) on the surface of T cells. The B7:CD28/CTLA4interaction has the effect of transmitting a signal to the T cell thatinduces the production of increased levels of cytokines, particularly ofinterleukin-2, which in turn stimulates the proliferation of the Tlymphocytes. In one embodiment, the CTLA4-immunoglobulin fusion proteinsof the invention have the functional property of being able to inhibitthe B7:CD28/CTLA4 interaction. Accordingly, other screening assays foridentifying a functional CTLA4-immunoglobulin fusion protein involveassaying for the ability of the fusion protein to inhibit synthesis ofcytokines, such as interleukin-2, interleukin-4 or other known orunknown novel cytokines and/or the ability to inhibit T cellproliferation by T cells which have received a primary activationsignal.

The ability of a CTLA4-immunoglobulin fusion protein of the invention toinhibit or block an interaction between a B7 family protein (e.g., B7-1or B7-2) with its receptor on T cells (e.g., CD28 and/or CTLA4) can beassessed in an in vitro T cell culture system by stimulating T cellswith a source of ligand (e.g., cells expressing B7-1 and/or B7-2 or asecreted form of B7-1 and/or B7-2) and a primary activation signal suchas antigen in association with Class II MHC (or alternatively, anti-CD3antibodies or phorbol ester) in the presence or absence of theCTLA4-immunoglobulin fusion protein. The culture supernatant is thenassayed for cytokine production, such as interleukin-2, gammainterferon, or other known or unknown cytokine. For example, any one ofseveral conventional assays for interleukin-2 can be employed, such asthe assay described in Proc. Natl. Acad. Sci. USA. 86:1333 (1989). Anassay kit for interferon production is also available from GenzymeCorporation (Cambridge, Mass.). T cell proliferation can be measured invitro by determining the amount of ³H-labeled thymidine incorporatedinto the replicating DNA of cultured cells. The rate and amount of DNAsynthesis and, in turn, the rate of cell division can thus bequantified. A lack of or reduction in the amount of cytokine productionand/or T cell proliferation by stimulated T cells upon culture with aCTLA4-immunoglobulin fusion protein of the invention indicates that thefusion protein is capable of inhibiting the delivery of a costimulatorysignal to the T cell by inhibiting an interaction between a CTLA4 ligand(e.g., B7-1 and/or B7-2) and a receptor therefor (e.g., CD28 and/orCTLA4).

The ability of the CTLAIg fusion protein to induce antigen-specific Tcell unresponsiveness or anergy can also be assessed using the in vitroT cell culture system described above. Following stimulation of the Tcells with a specific antigen bound to MHC molecules on an antigenpresenting cell surface and CTLA4 ligand (e.g., B7-1 on the antigenpresenting cell surface) in the presence of CTLA4-immunoglobulin fusionprotein, the T cells are subsequently restimulated with the antigen inthe absence of CTLA4-immunoglobulin fusion protein. A lack of cytokineproduction and/or T cell proliferation upon antigenic restimulation by Tcells previously treated with a CTLA4-immunoglobulin fusion protein ofthe invention indicates that the fusion protein has induced a state ofantigen-specific anergy or non-responsiveness in the T cells. See. e.g.,Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6586-6590; andSchwartz (1990) Science 248:1349-1356, for assay systems that can usedto examine T cell unresponsiveness in accordance with the presentinvention.

In yet another assay, the ability of a CTLA4-immunoglobulin fusionprotein of the invention to inhibit T cell dependent immune responses invitro is determined. The effect of a CTLA4-immunoglobulin fusion proteinon T_(h)-induced immunoglobulin production by B cells can be assessed bycontacting antigen-specific CD4⁺ T cells with syngeneic antigen-specificB cells, antigen and the CTLA4-immunoglobulin fusion protein. The cellculture supernatant is assayed for the production of immunoglobulin,such as IgG or IgM, using, for example, a solid phase ELISA or astandard plaque assay. Inhibition of B cell immunoglobulin production bytreatment of the culture with the CTLA4-immunoglobulin fusion proteinindicates that the protein is capable inhibiting T helper cell responsesand, consequently, T cell dependent B cell responses.

IV. Compositions of CTLA4-Immunoglobulin Fusion Proteins

The CTLA4-immunoglobulin fusion proteins of the invention can beincorporated into compositions suitable for administration to subjectsto thereby modulate immune responses or for other purposes (e.g.,antibody production). The CTLA4-immunoglobulin fusion protein in suchcompositions is in a biologically compatible form suitable forpharmaceutical administration in vivo. By “biologically compatible formsuitable for administration in vivo” is meant a form of the protein tobe administered in which any toxic effects are outweighed by thetherapeutic effects of the protein. The term subject is intended toinclude living organisms in which an immune response can be elicited,e.g., mammals. Examples of subjects include humans, monkeys, dogs, cats,mice, rats, and transgenic species thereof. Administration of aCTLA4-immunoglobulin fusion protein as described herein can be in anypharmacological form including a therapeutically active amount ofprotein and a pharmaceutically acceptable carrier. Administration of atherapeutically active amount of the therapeutic compositions of theinvention is defined as an amount effective, at dosages and for periodsof time necessary to achieve the desired result. For example, atherapeutically active amount of a CTLA4-immunoglobulin fusion proteinmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of protein to elicit a desiredresponse in the individual. Dosage regima may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active compound (e.g., CTLA4-immunoglobulin fusion protein) may beadministered in a convenient manner such as by injection (subcutaneous,intravenous, etc.), oral administration, inhalation, transdermalapplication, or rectal administration. Depending on the route ofadministration, the active compound may be coated in a material toprotect the compound from the action of enzymes, acids and other naturalconditions which may inactivate the compound.

To administer a CTLA4-immunoglobulin fusion protein by other thanparenteral administration, it may be necessary to coat the protein with,or co-administer the protein with, a material to prevent itsinactivation. For example, a CTLA4-immunoglobulin fusion protein may beadministered to an individual in an appropriate carrier, diluent oradjuvant, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous butter solutions. Adjuvant is used in its broadestsense and includes any immune stimulating compound, such as interferon.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polvalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., CTLA4-immunoglobulin fusion protein) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying, andfreeze-drying which yields a powder of the active ingredient (e.g.,peptide) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

When the active compound is suitably protected, as described above, theprotein may be orally administered, for example, with an inert diluentor an assimilable edible carrier. As used herein “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

V. Uses of CTLA4-Immunoglobulin Fusion Proteins Having Reduced IgCRegion-Mediated Biological Effector Functions

A. Immunomodulation

Given the role of CTLA4 ligands, such as B7-1 and B7-2, in T cellcostimulation and the structure and function of the CTLA4-immunoglobulinfusion proteins disclosed herein, the invention provides methods fordownregulating immune responses. The reduced IgC-region mediatedbiological effector functions exhibited by the mutatedCTLA4-immunoglobulin fusion proteins of the invention compared to aCTLA4-IgG1 fusion protein may result in more effective downregulation ofimmune responses in vivo without unwanted side effects (e.g., complementactivation, antibody-dependent cellular cytotoxicity, etc.) than if aCTLA4-IgG1 fusion protein were used. For example, improvements inmutated forms of CTLA4-immunoglobulin fusion proteins can be assessed bya variety of assays known to those skilled in the art, including variousanimal organ (heart, liver, kidney, bone marrow) transplantation modelsand in animal autoimmune disease models including, but not limited tolupus, multiple sclerosis, diabetes, and arthritis models.

Downregulation of an immune response by a CTLA4-immunoglobulin fusionprotein of the invention may be in the form of inhibiting or blocking animmune response already in progress or may involve preventing theinduction of an immune response. The functions of activated T cells,such as T cell proliferation and cytokine (e.g., IL-2) secretion, may beinhibited by suppressing T cell responses or by inducing specifictolerance in T cells, or both. Immunosuppression of T cell responses isgenerally an active process which requires continuous exposure of the Tcells to the suppressive agent and is often not antigen-specific.Tolerance, which involves inducing non-responsiveness or anergy in Tcells, is distinguishable from immunosuppression in that it is generallyantigen-specific and persists after exposure to the tolerizing agent hasceased. Operationally, T cell unresponsiveness or anergy can bedemonstrated by the lack of a T cell response upon reexposure tospecific antigen in the absence of the tolerizing agent.Immunosuppression and/or T cell unresponsiveness is achieved by blockingthe interaction of a CTLA4 ligand on an antigen presenting cell withCTLA4 itself and/or with another receptor for the CTLA4 ligand (e.g.,CD28) on the surface of a T cell, e.g., blocking the interaction of a B7family protein, such as B7-1 and/or B7-2, with a counter-receptor, suchas CD28 or CTLA4, on the surface of a T cell. The term “antigenpresenting cell” is intended to include B lymphocytes, professionalantigen presenting cells (e.g., monocytes, dendritic cells, Langerhancells) and others cells (e.g., keratinocytes, endothelial cells,astrocytes, fibroblasts, oligodendrocytes) which can present antigen toT cells. The CTLA4-immunoglobulin fusion proteins of the invention canbe used to inhibit CTLA4 ligand/receptor interactions in many clinicalsituations, as described further below.

1. Organ Transplantation/GVHD: Inhibition of T cell responses by aCTLA4-immunoglobulin fusion protein of the invention is useful insituations of cellular, tissue, skin and organ transplantation and inbone marrow transplantation (e.g., to inhibit graft-versus-host disease(GVHD)). For example, inhibition of T cell proliferation and/or cytokinesecretion may result in reduced tissue destruction in tissuetransplantation and induction of antigen-specific T cellunresponsiveness may result in long-term graft acceptance without theneed for generalized immunosuppression. Typically, in tissuetransplants, rejection of the graft is initiated through its recognitionas foreign by T cells, followed by an immune reaction that destroys thegraft. Administration of a CTLA4-immunoglobulin fusion protein of theinvention to a transplant recipient inhibits triggering of acostimulatory signal in alloantigen-specific T cells, thereby inhibitingT cell responses to alloantigens and, moreover, may inducegraft-specific T cell unresponsiveness in the recipient. The transplantrecipient can be treated with the CTLA4-immunoglobulin fusion proteinalone or together with one or more additional agents that inhibit thegeneration of stimulatory signals in the T cells (e.g., anti-B7-1 and/oranti-B7-2 antibodies, an anti-IL-2 receptor antibody) or induce generalimmunosuppression (e.g., cyclosporin A or FK506).

Use of a CTLA4-immunoglobulin fusion protein to inhibit triggering of acostimulatory signal in T cells can similarly be applied to thesituation of bone marrow transplantation to specifically inhibit theresponses of alloreactive T cells present in donor bone marrow and thusinhibit GVHD. A CTLA4-immunoglobulin fusion protein can be administeredto a bone marrow transplant recipient to inhibit the alloreactivity ofdonor T cells. Additionally or alternatively, donor T cells within thebone marrow graft can be tolerized to recipient alloantigens ex vivoprior to transplantation. For example, donor bone marrow can be culturedwith cells from the recipient (e.g., irradiated hematopoietic cells) inthe presence of a CTLA4-immunoglobulin fusion protein of the inventionprior to transplantation. Additional agents that inhibit the generationof stimulatory signals in the T cells (e.g., anti-B7-1 and/or anti-B7-2antibodies, an anti-IL-2R antibody etc., as described above) can beincluded in the culture. After transplantation, the recipient may befurther treated by in vivo administration of CTLA4-immunoglobulin (aloneor together with another agent(s) which inhibits the generation of acostimulatory signal in T cells in the recipient or inhibits theproduction or function of a T cell growth factor(s) (e.g., IL-2) in therecipient).

The efficacy of a particular CTLA4-immunoglobulin fusion protein ininhibiting organ transplant rejection or GVHD can be assessed usinganimal models that may be predictive of efficacy in humans. Given thehomology between CTLA4 molecules of different species, the functionallyimportant aspects of CTLA4 are believed to be conserved structurallyamong species thus allowing animal systems to be used as models forefficacy in humans. Examples of appropriate systems which can be usedinclude allogeneic cardiac grafts in rats and xenogeneic pancreaticislet cell grafts in mice, both of which have been used to examine theimmunosuppressive effects of CTLA4-IgG1 fusion proteins in vivo asdescribed in Lenschow et al., Science, 257: 789-792 (1992) and Turka etal., Proc. Natl. Acad. Sci. USA. 89: 11102-11105 (1992). In addition,murine models of GVHD (see Paul ed., Fundamental Immunology, RavenPress, New York, 1989, pp. 846-847) can be used to determine the effectof treatment with a CTLA4-immunoglobulin fusion protein of the inventionon the development of that disease.

As an illustrative embodiment, a CTLA4-immunoglobulin fusion protein ofthe invention can be used in a rat model of organ transplantation toascertain the ability of the fusion protein to inhibit alloantigenresponses in vivo. Recipient Lewis rats receive a Brown-Norway ratstrain cardiac allograft which is anastamosed to vessels in the neck asdescribed in Bolling, S. F. et al., Transplant, 453:283-286 (1992).Grafts are monitored for mechanical function by palpation and forelectrophysiologic function by electrocardiogram. Graft rejection issaid to occur on the last day of palpable contractile function. As aninitial test, animals are treated with daily injections of aCTLA4-immunoglobulin fusion protein of interest, an isotype-matchedcontrol Ig fusion protein and/or CTLA4-IgG1 (for comparison purposes)for 7 days. Fusion proteins are administered at a dosage range betweenapproximately 0.015 mg/day and 0.5 mg/day. Untreated Lewis ratstypically reject heterotopic Brown-Norway allografts in about 7 days.The rejection of allografts by fusion protein-treated animals isassessed in comparison to untreated controls.

An untreated animal and a fusion protein-treated animal are sacrificedfor histological examination. Cardiac allografts are removed from theuntreated animal and the treated animal four days after transplantation.Allografts are fixed in formalin, and tissue sections are stained withhematoxylin-eosin. The heart tissue of the untreated and treated animalsis examined histologically for severe acute cellular rejection includinga prominent interstitial mononuclear cell infiltrate with edemaformation, myocyte destruction, and infiltration of arterlolar walls.The effectiveness of the fusion protein treatment in inhibiting graftrejection is supported by a lack of an acute cellular rejection in theheart tissue of the fusion protein treated animals.

To determine whether fusion protein therapy establishes long term graftacceptance that persists following treatment, animals treated for 7 dayswith daily injections of fusion protein are observed without additionaltherapy until cessation of graft function. Graft survival is assesseddaily as described above. Allografts are examined histologically fromanimals in which the graft stops functioning as described above.Induction of graft tolerance by fusion protein treatment is indicated bythe continued functioning of the graft following the cessation oftreatment with the fusion protein.

After prolonged graft acceptance, a fusion protein-treated animal can besacrificed and the lymphocytes from the recipient can be tested fortheir functional responses. These responses are compared with those oflymphocytes from a control (non-transplanted) Lewis rat, and results arenormalized as a percentage of the control response. The T cellproliferative response to ConA and to cells from a Brown-Norway rat anda third party ACI rat can be examined. Additionally, the thymus andspleen from the untreated and treated animals can be compared in size,cell number and cell type (e.g. by flow cytometic analyses of thymus,lymph nodes and spleen cells). Specific nonresponsiveness in the treatedanimals to alloantigens, as a result of specific clonal deletion ofalloreactive cells, is indicated by the ability of the T cells torespond to ConA and third party stimulators (e.g., ACI rat cells) butnot to Brown-Norway rat cells. Prolonged acceptance of allografts,including continued graft acceptance following CTLA4-immunoglobulintreatment, in this model system may be predictive of the therapeuticefficacy of the CTLA4-immunoglobulin fusion proteins of the invention inhuman transplant situations.

2. Autoimmune Diseases: Inhibition of T cell responses by aCTLA4-immunoglobulin fusion protein of the invention may also betherapeutically useful for treating autoimmune diseases. Many autoimmunedisorders are the result of inappropriate activation of T cells that arereactive against self tissue (i.e., reactive against autoantigens) andwhich promote the production of cytokines and autoantibodies involved inthe pathology of the diseases. Preventing the activation of autoreactiveT cells thus may reduce or eliminate disease symptoms. Administration ofa CTLA4-immunoglobulin fusion protein of the invention to a subjectsuffering from or susceptible to an autoimmune disorder may inhibitautoantigen-specific T cell responses and induce autoantigen-specific Tcell unresponsiveness, thereby inhibiting or preventing production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process.

To treat an autoimmune disorder, a CTLA4-immunoglobulin fusion proteinof the invention is administered to a subject in need of treatment. Forautoimmune disorders with a known autoantigen, it may be desirable tocoadminister the autoantigen with the CTLA4-immunoglobulin to thesubject. This method can be used to treat a variety of autoimmunediseases and disorders having an autoimmune component, includingdiabetes mellitus, arthritis (including rheumatoid arthritis, juvenilerheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiplesclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmunethyroiditis, dermatitis (including atopic dermatitis and eczematousdermatitis), psoriasis, Sjögren's Syndrome, includingkeratoconjunctivitis sicca secondary to Sjögren's Syndrome, alopeciaareata, allergic responses due to arthropod bite reactions, Crohn'sdisease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis,ulcerative colitis, asthma, allergic asthma, cutaneous lupuserythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primarybiliary cirrhosis, uveitis posterior, and interstitial lung fibrosis.

The efficacy of a CTLA4-immunoglobulin fusion protein of the inventionin preventing or alleviating autoimmune disorders can be determinedusing a number of well-characterized animal models of human autoimmunediseases. Examples include murine experimental autoimmune encephalitis,systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice,murine autoimmune collagen arthritis, diabetes mellitus in NOD mice andBB rats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Experimental Autoimmune Encephalomyelitis (EAE) is a rodent and primatemodel for multiple sclerosis. In an illustrative embodiment utilizingthe passive EAE model, donor mice are immunized with 0.4 mg Myelin BasicProtein (MBP) in Complete Freund's Adjuvant (CFA), divided over fourquadrants. The draining axillary and inguinal lymph nodes are removedeleven days later. Lymph node cells (4×10⁶/ml) are plated in 2 mlcultures in 24 well plates, in the presence of 25 μg/ml MBP. After fourdays in culture, 30×10⁶ of the treated cells are injected into the tailvein of each naive, syngeneic recipient mouse.

The recipient mice develop a remitting, relapsing disease and areevaluated utilizing the following criteria:

0 normal, healthy

1 limp tail, incontinence; occasionally the first sign of the disease isa “tilt”

2 hind limb weakness, clumsiness

3 mild paraparesis

4 severe paraparesis

5 quadriplegia

6 death

Using the passive model of EAE, the effect of CTLA4-immunoglobulintreatment of the donor cells on resulting disease severity in arecipient animal is tested in mice (e.g., the PLSJLF1/J strain). Cultureof lymph node cells in vitro with MBP is performed either in thepresence or the absence of about 30 μg/ml of a CTLA4-immunoglobulinfusion protein of the invention, an isotype matched control Ig fusionprotein or CTLA4IgG1 (for comparison purposes). The treated cells arethen introduced into a syngeneic recipient mouse. The effect of fusionprotein treatment of donor cells on the severity of the recipient'sfirst episode of disease as compared to mice receiving untreated cellscan be determined using the above-described criteria to assess diseaseseverity. In addition, ensuing relapses in the mice receiving fusionprotein-treated cells versus untreated cells can be assessed using theabove-described criteria.

The effect of treating both the donor mice and the cultured donor cellswith CTLA4-immunoglobulin on the clinical disease severity in therecipient can further be assessed. In these experiments, donor mice(e.g., of the SJL/J strain) immunized with MBP are given either 100 μgof CTLA4-immunoglobulin fusion protein, an isotype matched control Igfusion protein or CTLA4-IgG1 (for comparison) intraperitoneally each dayfor eleven days. Cells are then isolated from lymph nodes of thesedonors and cultured with MBP in vitro in the presence of either 30 μg/mlof CTLA4-immunoglobulin fusion protein or control fusion proteins. Thetreated cells are then introduced into a syngeneic recipient. The effectof fusion protein treatment on the severity of the ensuing disease inthe recipient is then assessed using the above-described criteria.

Studies using a direct (active) model of EAE can also conducted. Inthese experiments, a CTLA4-immunoglobulin fusion protein of theinvention or control fusion protein is directly administered to miceimmunized with MBP and treated with pertussis toxin (PT). Mice (e.g.,the PLSJLFI/J strain) are immunized with MBP on day 0, injected with PTintravenously on days 0 and 2, and given either a CTLA4-immunoglobulinfusion protein of the invention or a control fusion protein on days 0 to7. The effect of direct fusion protein treatment of the MBP-immunizedmice on the severity of the ensuing disease is then assessed using theabove-described criteria. A reduced severity in disease symptoms in thepassive and/or active EAE model as a result of CTLA4-immunoglobulintreatment may be predictive of the therapeutic efficacy of theCTLA4-immunoglobulin fusion proteins of the invention in humanautoimmune diseases.

3. Allergy:

The IgE antibody response in atopic allergy is highly T cell dependentand, thus, inhibition of CTLA4 ligand/receptor induced T cell activationmay be useful therapeutically in the treatment of allergy and allergicreactions. A CTLA4-immunoglobulin fusion protein of the invention can beadministered to an allergic subject to inhibit T cell mediated allergicresponses in the subject. Inhibition of costimulation of T cells throughinhibition of a CTLA4 ligand/receptor interaction may be accompanied byexposure to allergen in conjunction with appropriate MHC molecules.Exposure to the allergen may be environmental or may involveadministering the allergen to the subject. Allergic reactions may besystemic or local in nature, depending on the route of entry of theallergen and the pattern of deposition of IgE on mast cells orbasophils. Thus, it may be necessary to inhibit T cell mediated allergicresponses locally or systemically by proper administration of aCTLA4-immunoglobulin fusion protein of the invention. For example, inone embodiment, a CTLA4-immunoglobulin fusion protein of the inventionand an allergen are coadminstered subcutaneously to an allergic subject.

4. Virally Infected or Malignant T Cells: Inhibition of T cellactivation through blockage of the interaction of a CTLA4 ligand with areceptor therefor on T cells may also be important therapeutically inviral infections of T cells. For example, in the acquired immunedeficiency syndrome (AIDS), viral replication is stimulated by T cellactivation. Blocking a CTLA4 ligand/receptor interaction, such as theinteraction of B7-1 and/or B7-2 with CD28 and/or CTLA4 could lead to alower level of viral replication and thereby ameliorate the course ofAIDS. Surprisingly, HTLV-I infected T cells express B7-1 and B7-2. Thisexpression may be important in the growth of HTLV-I infected T cells andthe blockage of B7-1 function together with the function of B7-2 with aCTLA4-immunoglobulin fusion protein, possibly in conjunction withanother blocking reagent (such as an anti-B7-2 blocking antibody or aCD28Ig fusion protein) may slow the growth of HTLV-I induced leukemias.In addition, some tumor cells are responsive to cytokines and theinhibition of T cell activation and cytokine production could help toinhibit the growth of these types of cancer cells.

5. Antigen-Specific T Cell Unresponsiveness: The methods of theinvention for inhibiting T cell responses can essentially be applied toany antigen (e.g., protein) to clonally delete T cells responsive tothat antigen in a subject. For example, in one study, administration ofa CTLA4-IgG1 fusion protein to mice in vivo suppressed primary andsecondary T cell-dependent antibody responses to antigen (Linsley P. S.,et al. (1992) Science 257, 792-795). Thus, a subject treated with amolecule capable of inducing a T cell response can be treated withCTLA4-immunoglobulin fusion protein to inhibit T cell responses to themolecule. This basic approach has widespread application as an adjunctto therapies which utilize a potentially immunogenic molecule fortherapeutic purposes. For example, an increasing number of therapeuticapproaches utilize a proteinaceous molecule, such as an antibody, fusionprotein or the like, for treatment of a clinical disorder. A limitationto the use of such molecules therapeutically is that they can elicit animmune response directed against the therapeutic molecule in the subjectbeing treated (e.g., the efficacy of murine monoclonal antibodies inhuman subjects is hindered by the induction of an immune responseagainst the antobodies in the human subject). Administration of aCTLA4-immunoglobulin fusion protein to inhibit antigen-specific T cellresponses can be applied to these therapeutic situations to enable longterm usage of the therapeutic molecule in the subject withoutelicitation of an immune response. For example, a therapeutic antibody(e.g., murine mAb) is administered to a subject (e.g., human), whichtypically activates T cells specific for the antibody in the subject. Toinhibit the T cell response against the therapeutic antibody, thetherapeutic antibody is administered to the subject together with aCTLA4-immunoglobulin fusion protein of the invention.

When used therapeutically, a CTLA4-immunoglobulin fusion protein of theinvention can be used alone or in conjunction with one or more otherreagents that influence immune responses. A CTLA4-immunoglobulin fusionprotein and another immunomodulating reagent can be combined as a singlecomposition or administered separately (simultaneously or sequentially)to downregulate T cell mediated immune responses in a subject. Examplesof other immunomodulating reagents include blocking antibodies, e.g.,against B7-1, B7-2 or other B cell surface antigens or cytokines, otherfusion proteins, e.g., CD28Ig, or immunosuppressive drugs, e.g.,cyclosporine A or FK506.

The CTLA4-immunoglobulin fusion proteins of the invention may also beuseful in the construction of therapeutic agents which block immune cellfunction by destruction of the cell. For example, by linking aCTLA4-immunoglobulin fusion protein to a toxin such as ricin ordiptheria toxin, an agent capable of preventing immune cell activationwould be made. Infusion of one or a combination of immunotoxins into apatient would result in the death of immune cells, particularly ofactivated B cells that express higher amounts of B7-1 and/or B7-2.

B. Screening Assays

Another application of the CTLA4-immunoglobulin fusion proteins of theinvention is the use the protein in screening assays to discover as yetundefined molecules which inhibit an interaction between CTLA4 and aCTLA4 ligand, such as B7-1 or B7-2. For example, theCTLA4-immunoglobulin fusion protein can be used in a solid-phase bindingassay in which panels of molecules are tested. In one embodiment, thescreening method of the invention involves contacting aCTLA4-immunoglobulin fusion protein of the invention with a CTLA4 ligandand a molecule to be tested. Either the CTLA4-immunoglobulin fusionprotein or the CTLA4 ligand is labeled with a detectable substance, suchas a radiolabel or biotin, which allows for detection and quantitationof the amount of binding of CTLA4-immunoglobulin to the CTLA4 ligand.After allowing CTLA4-immunoglobulin and the CTLA4 ligand to interact inthe presence of the molecule to be tested, unbound labeledCTLA4-immunoglobulin fusion protein or unbound labeled CTLA4 ligand isremoved and the amount of CTLA4-immunoglobulin fusion protein bound tothe CTLA4 ligand is determined. A reduced amount of binding ofCTLA4-immunoglobulin fusion protein to the CTLA4 ligand in the presenceof the molecule tested relative to the amount of binding in the absenceof the molecule is indicative of an ability of the molecule to inhibitbinding of CTLA4 to the CTLA4 ligand. Suitable CTLA4 ligands for use inthe screening assay include B7-1 or B7-2 (e.g., B7-1Ig or B7-2Ig fusionproteins can be used). Preferably, either the unlabeledCTLA4-immunoglobulin fusion protein or the unlabeled CTLA4 ligand isimmobilized on a solid phase support, such as a polystyrene plate orbead, to facilitate removal of the unbound labeled protein from thebound labeled protein.

C. Antibody Production

The CTLA4-immunoglobulin fusion proteins produced from the nucleic acidmolecules of the invention can also be used to produce antibodiesspecifically reactive with the fusion protein and in particular with theCTLA4 moiety thereof (i.e., anti-CTLA4 antibodies). For example, byimmunization with a CTLA4-immunoglobulin fusion protein, anti-CTLA4polyclonal antisera or monoclonal antibodies can be made using standardmethods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunizedwith an immunogenic form of the fusion protein which elicits an antibodyresponse in the mammal. Techniques for conferring immunogenicity on aprotein include conjugation to carriers or other techniques well knownin the art. For example, the protein can be administered in the presenceof adjuvant. The progress of immunization can be monitored by detectionof antibody titers in plasma or serum. Standard ELISA or otherimmunoassay can be used with the immunogen as antigen to assess thelevels of antibodies. An ELISA or other immunoassay which distinguishesantibodies reactive with the CTLA4 portion of the fusion protein fromthose which react with the IgC region are preferred (e.g., theextracellular domain of CTLA4 alone can be used in a standard ELISA todetect anti-CTLA4 antibodies).

Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera. To produce monoclonalantibodies, antibody producing cells (lymphocytes) can be harvested froman immunized animal and fused with myeloma cells by standard somaticcell fusion procedures thus immortalizing these cells and yieldinghybridoma cells. Such techniques are well known in the art. Examplesinclude the hybridoma technique originally developed by Kohler andMilstein (Nature (1975) 256:495-497) as well as other techniques such asthe human B-cell hybridoma technique (Kozbar et al., Immunol. Today(1983) 4:72), the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985)(Allen R. Bliss, Inc., pages 77-96), and screening of combinatorialantibody libraries (Huse et al., Science (1989) 246:1275). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with CTLA4 and monoclonal antibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with a CTLA4-immunoglobulinfusion protein as described herein. Antibodies can be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments. The term “antibody” is further intended toinclude bispecific and chimeric molecules having ananti-CTLA4-immunoglobulin fusion protein portion, chimeric antibodyderivatives, i.e., antibody molecules that combine a non-human animalvariable region and a human constant region, and humanized antibodies inwhich parts of the variable regions, especially the conserved frameworkregions of the antigen-binding domain, are of human origin and only thehypervariable regions are of non-human origin. Techniques for preparingchimeric or humanized antibodies are well known in the art (see e.g.,Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1985); Takeda etal., Nature 314:452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567;Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP171496; European Patent Publication 0173494, UnitedKingdom Patent GB 2177096B, Teng et al., Proc. Natl. Acad. Sci. U.S.A.,80:7308-7312 (1983); Kozbor et al., Immunology Today, 4:7279 (1983);Olsson et al., Meth. Enzymol., 92:3-16 (1982); PCT PublicationWO92/06193 and EP 0239400). Another method of generating specificantibodies, or antibody fragments, reactive against aCTLA4-immunoglobulin fusion protein is to screen expression librariesencoding immunoglobulin genes, or portions thereof, expressed inbacteria with a fusion protein produced from the nucleic acid moleculesof the invention. For example, complete Fab fragments, V_(H) regions,F_(V) regions and single chain F_(V) regions can be expressed inbacteria using phage expression libraries. See for example Ward et al.,Nature, 341: 544-546: (1989); Huse et al., Science, 246: 1275-1281(1989); and McCafferty et al., Nature, 348: 552-554 (1990). Screeningsuch libraries with, for example, a CTLA4-immunoglobulin fusion proteincan identify immunoglobin fragments reactive with the protein, inparticular the CTLA4 portion thereof.

An anti-CTLA4 antibody generated using the CTLA4-immunoglobulin fusionproteins described herein can be used therapeutically to inhibit immunecell activation through blocking receptor:ligand interactions necessaryfor stimulation of the cell. These so-called “blocking antibodies” canbe identified by their ability to inhibit T cell proliferation and/orcytokine production when added to an in vitro costimulation assay asdescribed herein. The ability of blocking antibodies to inhibit T cellfunctions may result in immunosuppression and/or tolerance when theseantibodies are administered in vivo.

D. Protein Purification

The CTLA4-immunoglobulin fusion proteins of the invention can be used toisolate CTLA4 ligands from cell extracts or other preparations. Forexample, a CTLA4-immunoglobulin fusion protein can be used toimmunoprecipitate B7-1, B7-2 or an as yet unknown CTLA4 ligand from awhole cell, cytosolic or membrane protein extract prepared from B cellsor other antigen presenting cell using standard techniques.Additionally, anti-CTLA4 polyclonal or monoclonal antibodies prepared asdescribed herein using a CTLA4-immunoglobulin fusion protein as animmunogen can be used to isolate the native CTLA4 antigen from cells.For example, antibodies reactive with the CTLA4 portion of theCTLA4-immunoglobulin fusion protein can be used to isolate thenaturally-occurring or native form of CTLA4 from activated T lymphocytesby immunoaffinity chromatography using standard techniques.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patent applications cited throughout this application arehereby incorporated by reference.

EXAMPLE 1 Preparation of CTLA4-Immunoglobulin Fusion Proteins withReduced Effector Function

The extracellular portion of the T cell surface receptor CTLA4 wasprepared as a fusion protein coupled to an immunoglobulin constantregion. The immunoglobulin constant region was genetically modified toreduce or eliminate effector activity inherent in the immunoglobulinstructure. Briefly, DNA encoding the extracellular portion of CTLA4 wasjoined to DNA encoding the hinge, CH2 and CH3 regions of human IgCγ1 orIgCγ4 modified by directed mutagenesis. This was accomplished asfollows:

Preparation of Gene Fusions

DNA fragments corresponding to the DNA sequences of interest wereprepared by polymerase chain reaction (PCR) using primer pairs describedbelow. In general, PCR reactions were prepared in 100 μl final volumecomposed of Taq polymerase buffer (Gene Amp PCR Kit, Perkin-Elmer/Cetus,Norwalk, Conn.) containing primers (1 μM each). dNTPs (200 μM each), 1ng of template DNA, and Taq polymerase (Saiki, R. K., et al. (1988)Science 239:487-491). PCR DNA amplifications were run on a thermocycler(Ericomp, San Diego, Calif.) for 25 to 30 cycles each composed of adenaturation step (1 minute at 94° C.), a renaturation step (30 secondsat 54° C.), and a chain elongation step (1 minute at 72° C.).

To create gene fusions encoding hybrid proteins, “zip up” PCR was used.This procedure is diagrammed schematically in FIG. 1. A first set offorward (A) and reverse (C) primers was used to amplify the first genesegment of the gene fusion. A second set of forward (B) and reverse (D)primers was used to amplify the second gene segment of the gene fusion.Primers B and C were designed such that they contained complimentarysequences capable of annealing. The PCR products amplified by primersA+C and B+D are combined, annealed and extended (“zipped up”). Thefull-length gene fusion was then amplified in a third PCR reaction usingthe “zip up” fragment as the template and primers A and D as the forwardand reverse primers, respectively.

The structure of each CTLA4 genetic fusion consisted of a signalsequence, to facilitate secretion, coupled to the extracellular domainof CTLA4 and the hinge, CH2 and CH3 domains of human IgCγ1 or IgCγ4. TheIgCγ1 and IgCγ4 sequences were modified to contain nucleotide changeswithin the hinge region to replace cysteine residues available fordisulfide bond formation and to contain nucleotide changes in the CH2domain to replace amino acids thought to be required for IgC binding toFc receptors and complement activation. The hinge region and CH2 domainamino acid mutations introduced into IgCγ1 and IgCγ4 are illustrated inFIGS. 2A and 2B, respectively.

A. Construction of CTLA4-Ig Fusion Genes

1. Preparation of the Signal Sequence Gene Segment

PCR amplification was used to generate an immunoglobulin signal sequencesuitable for secretion of the CTLA4-Ig fusion protein from mammaliancells. The Ig signal sequence was prepared from a plasmid containing themurine IgG heavy chain gene (described in Orlandi, R., et al. (1989)Proc. Natl. Acad. Sci. USA 86:3833-3837) using the oligonucleotide5′CATTCTAGAACCTCGACAAGCTTGAGATCACAGTTCTCTCTAC-3′ (SEQ ID NO: 1) as theforward primer and the oligonucleotide 5′CAGCAGGCTGGGCCACGTGCATTGCGGAGTGGACACCTGTGGAGAG-3′ (SEQ ID NO: 2) as the reverse PCR primer. Theforward PCR primer (SEQ ID NO: 1) contains recognition sequences forrestriction enzymes XbaI and HindIII and is homologous to sequences 5′to the initiating methionine of the Ig signal sequence. The reverse PCRprimer (SEQ ID NO: 2) is composed of sequences derived from the 5′ endof the extracellular domain of CTLA4 and the 3′ end of the Ig signalsequence. PCR amplification of the murine Ig signal template DNA usingthese primers resulted in a 233 bp product which is composed of XbaI andHindIII restriction sites followed by the sequence of the Ig signalregion fused to the first 25 nt of the coding sequence of theextracellular domain of CTLA4. The junction between the signal sequenceand CTLA4 is such that protein translation beginning at the signalsequence will continue into and through CTLA4 in the correct readingframe.

2. Preparation of the CTLA4 Gene Segment

The extracellular domain of the CTLA4 gene was prepared by PCRamplification of plasmid phCTLA4. This plasmid contained the sequencescorresponding to the human CTLA4 cDNA (see Darivach, P., et al., (1988)Eur. J. Immunol. 18:1901 1905; Harper, K., et al., (1991) J. Immunol.147: 1047-1044) inserted into the multiple cloning site of vectorpBluescript (Stratagene, La Jolla, Calif.) and served as the templatefor a PCR amplification using the oligonucleotide5′-CTCTCCACAGGTGTCCACTCCGCAATGCACGTGG CCCAGCCTGCTG-3′ (SEQ ID NO: 3) asthe forward PCR primer and the oligonucleotide5′-TGTGTGTGGAATTCTCATTACTGATCAGAATCTGGGCACGGTTCTG-3′ (SEQ ID NO: 4) asthe reverse PCR primer. The forward PCR primer (SEQ ID NO: 3) wascomposed of sequences derived from the 3′ end of the Ig signal sequenceand the 5′ end of the extracellular domain of CTLA4. This PCR primer isthe complementary to murine Ig signal reverse PCR primer (SEQ ID NO: 2).The reverse PCR primer (SEQ ID NO: 4) was homologous to the 3′ end ofthe extracellular domain of CTLA4, added a BclI restriction site and anadditional G nucleotide at the end of the extracellular domain. Thiscreated a unique BclI restriction site and added a glutamine codon tothe C-terminus of the extracellular domain. The final PCR product was413 bp.

3. Fusion of the Immunoglobulin Signal Sequence and CTLA4 Gene Segments

The PCR fragments containing the signal and CTLA4 sequences were joinedtogether via a third PCR reaction. Both PCR fragments (1 ng each) weremixed together along with the Ig signal forward PCR primer (SEQ IDNO: 1) and the CTLA4 reverse PCR primer (SEQ ID NO: 4) and PCR amplifiedas described. In this reaction, the 3′ end of the Ig signal fragmenthybridizes with the 5′ end of the CTLA4 fragment and the two strands areextended to yield a full length 600 bp fragment. Subsequent PCRamplification of this fragment using forward (SEQ ID NO: 1) and reverse(SEQ ID NO: 4) yielded sufficient amounts of the signal-CTLA4 genefusion fragment for cloning. This fragment contains a 5′ XbaI and a 3′BclI restriction sites flanking the Ig signalCTLA4 gene fusion segmentfor subsequent cloning.

4. Cloning of Immunoylobulin Constant Domain Gene Segments

Plasmid pSP72IgG1 was prepared by cloning the 2000 bp segment of humanIgG1 heavy chain genomic DNA (Ellison, J. W., et al., (1982) Nucl.Acids. Res. 10:4071-4079) into the multiple cloning site of cloningvector pSP72 (Promega, Madison, Wis.). Plasmid pSP72IgG1 containedgenomic DNA encoding the CH1, hinge, CH2 and CH3 domain of the heavychain human IgCγ1 gene. PCR primers designed to amplify thehinge-CH2-CH3 portion of the heavy chain along with the interveninggenomic DNA were prepared as follows. The forward PCR primer,5′-GCATTTTAAGCTTTTTCCTGATCAGGAGCCCAAATCTTCTGACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3′ (SEQ ID NO: 5),contained HindIII and BclI restriction sites and was homologous to thehinge domain sequence except for five nucleotide substitutions whichchanged the three cysteine residues to serines. The reverse PCR primer,5′-TAATACGACTCACTATAGGG-3′ (SEQ ID NO: 6), was identical to thecommercially available T7 primer (Promega, Madison, Wis.). Amplificationwith these primers yielded a 1050 bp fragment bounded on the 5′ end byHindIII and BclI restriction sites and on the 3′ end by BamHI, SmaI,KpnI, SacI, EcoRI, ClaI, EcoR5 and BglII restriction sites. Thisfragment contained the IgCγ1 hinge domain in which the three cysteinecodons had been replaced by serine codons followed by an intron, the CH2domain, an intron, the CH3 domain and additional 3′ sequences. After PCRamplification, the DNA fragment was digested with HindIII and EcoRI andcloned into expression vector pNRDSH (Repligen; Cambridge, Mass.(diagrammed in FIG. 3)) digested with the same restriction enzymes. Thiscreated plasmid pNRDSH/IgG1.

A similar PCR based strategy was used to clone the hinge-CH2-CH3 domainsof human IgCγ4 constant regions. A plasmid, p428D (Medical ResearchCouncil, London, England) containing the complete IgCγ4 heavy chaingenomic sequence (Ellison. J., et al., (1981) DNA 1: 11-18) was used asa template for PCR amplification using oligonucleotide5′GAGCATTTTCCTGATCAGGAGTCCAAATATGGTCCCCCACCCC ATCATCCCCAGGTAAGCCAACCC-3′(SEQ ID NO: 7) as the forward PCR primer and oligonucleotide5′GCAGAGGAATTCGAGCTCGGTACCCGGGGATCCCCAGTGTGGGGACAGTGGGACCCGCTCTGCCTCCC-3′ (SEQ ID NO: 8) as the reverse PCR primer.The forward PCR primer (SEQ ID NO: 7) contains a BclI restriction sitefollowed by the coding sequence for the hinge domain of IgCγ4.Nucleotide substitutions have been made in the hinge region to replacethe cysteines residues with serines. The reverse PCR primer (SEQ ID NO:8) contains a PspAI restriction site. PCR amplification with theseprimers results in a 1179 bp DNA fragment. The PCR product was digestedwith BclI and PspAI and ligated to pNRDSH/IgG1 digested with the samerestriction enzymes to yield plasmid pNRDSH/IgG4. In this reaction, theIgCγ4 domain replaced the IgCγ1 domain present in pNRDSH/IgG1.

5. Modification of Immunoglobulin Constant Domain Gene Segments

Modification of the CH2 domain in IgC to replace amino acids thought tobe involved in binding to Fc receptor was accomplished as follows.Plasmid pNRDSH/IgG1 served as template for modifications of the IgCγ1CH2 domain and plasmid pNRDSH/IgG4 served as template for modificationsof the IgCγ4 CH2 domain. Plasmid pNRDSH/IgG1 was PCR amplified using aforward PCR primer (SEQ ID NO: 5) and oligonucleotide 5′-GGGTTTTGGGGGGAAGAGGAAGACTGACGGTGCCCCC TCGGCTTCAGGTGCTGAGGAAG-3′ (SEQ ID NO: 9)as the reverse PCR primer. The forward PCR primer (SEQ ID NO: 5) hasbeen previously described and the reverse PCR primer (SEQ ID NO: 9) washomologous to the amino terminal portion of the CH2 domain of IgG1except for five nucleotide substitutions designed to change amino acids234, 235, and 237 from Leu to Ala, Leu to Glu, and Gly to Ala,respectively (Canfield, S. M. and Morrison, S. L. (1991) J. Exp. Med.173: 1483-1491; see FIG. 2A). Amplification with these PCR primers willyield a 239 bp DNA fragment consisting of a modified hinge domain, anintron and modified portion of the CH2 domain.

Plasmid pNRDSH/IgG1 was also PCR amplified with the oligonucleotide5′-CATCTCTTCCTCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCC CC-3′ (SEQ IDNO: 10) as the forward primer and oligonucleotide (SEQ ID NO: 6) as thereverse PCR primer. The forward PCR primer (SEQ ID NO: 10) iscomplementary to primer (SEQ ID NO: 9) and contains the fivecomplementary nucleotide changes necessary for the CH2 amino acidreplacements. The reverse PCR primer (SEQ ID NO: 6) has been previouslydescribed. Amplification with these primers yields a 875 bp fragmentconsisting of the modified portion of the CH2 domain, an intron, the CH3domain, and 3′ additional sequences.

The complete IgCγ1 segment consisting of modified hinge domain, modifiedCH2 domain and CH3 domain was prepared by an additional PCR reaction.The purified products of the two PCR reactions above were mixed,denatured (95° C., 1 minute) and then renatured (54° C., 30 seconds) toallow complementary ends of the two fragments to anneal. The strandswere filled in using dNTP and Taq polymerase and the entire fragmentamplified using forward PCR primer (SEQ ID NO: 5) and reverse PCR primer(SEQ ID NO: 6). The resulting fragment of 1050 bp was purified, digestedwith HindIII and EcoRI and ligated to pNRDSH previously digested withthe same restriction enzymes to yield plasmid pNRDSH/IgG1m.

Two amino acids at immunoglobulin positions 235 and 237 were changedfrom Leu to Glu and Gly to Ala, respectively within the IgCγ4 CH2 domainto eliminate Fc receptor binding (see FIG. 2B). Plasmid pNRDSH/IgG4 wasPCR amplified using the forward primer (SEQ ID NO: 7) and theoligonucleotide 5′CGCACGTGACCTCAGGGGTCCGGGAGATCATGAGAGTGTCCTTGGGTTTTGGGGGGAACAGGAAGACTGATGGTGCCCCCTCGAACTCAGGTGCTGAGG-3′ (SEQ ID NO: 11) as the reverse primer.The forward primer has been previously described and the reverse primerwas homologous to the amino terminal portion of the CH2 domain, exceptfor three nucleotide substitutions designed to replace the amino acidsdescribed above. This primer also contained a PmlI restriction site forsubsequent cloning. Amplification with these primers yields a 265 bpfragment composed of the modified hinge region, and intron, and themodified 5′ portion of the CH2 domain.

Plasmid pNRDSH/IgG4 was also PCR amplified with the oligonucleotide5′-CCTCAGCACCTGAGTTCGAGGGGGCACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG-3′ (SEQ ID NO: 12) asthe forward primer and oligonucleotide (SEQ ID NO: 8) as the reverse PCRprimer. The forward PCR primer (SEQ ID NO: 12) is complementary toprimer (SEQ ID NO: 11) and contains the three complementary nucleotidechanges necessary for the CH2 amino acid replacements. The reverse PCRprimer (SEQ ID NO: 8) has been previously described. Amplification withthese primers yields a 1012 bp fragment consisting of the modifiedportion of the CH2 domain, an intron, the CH3 domain, and 3′ additionalsequences.

The complete IgCγ4 segment consisting of modified hinge domain, modifiedCH2 domain and CH3 domain was prepared by an additional PCR reaction.The purified products of the two PCR reactions above were mixed,denatured (95° C., 1 minute) and then renatured (54° C., 30 seconds) toallow complementary ends of the two fragments to anneal. The strandswere filled in using dNTP and Taq polymerase and the entire fragmentamplified using forward PCR primer (SEQ ID NO: 7) and reverse PCR primer(SEQ ID NO: 8). The resulting fragment of 1179 bp was purified digestedwith BclI and PspAI and ligated to pNRDSH previously digested with thesame restriction enzymes to yield plasmid pNRDSH/IgG4m.

6. Assembly of CTLA4-Immunoglobulin Fusion Genes

The PCR fragment corresponding to the Ig signal-CTLA4 gene fusionprepared as described above (sections 1-3) was digested with HindIII andBclI restriction enzymes and ligated to pNRDSH/IgG1, pNRDSH/IgG1m,pNRDSH/IgG4, and pNRDSH/IgG4m previously digested with the samerestriction enzymes to create expression plasmids in which thesignal-CTLA4-IgG gene fusion segment is placed under the control of theCMV promoter. The ligated plasmids were transformed into E. coli JM109using CaCl₂ competent cells and transformants were selected on L-agarcontaining ampicillin (50 μg/ml; as described in Molecular Cloning: ALaboratory Manual (1982) Eds. Maniatis, T., Fritsch, E. E., andSambrook, J. Cold Spring Harbor Laboratory). Plasmids isolated from thetransformed E. coli were analyzed by restriction enzyme digestion.Plasmids with the expected restriction pattern were sequenced to verifyall portions of the signal-CTLA4-IgG gene fusion segments. The finalplasmids were named pNRDSH/sigCTLA4-IgG1, pNRDSH/sigCTLA4-IgG1m,pNRDSH/sigCTLA4-IgG4 and pNRDSH/sigCTLA4-IgG4m. The signal-CTLA4-IgGgene fusion segments from each of these constructs were also transferredto the pEE12 expression vector (Biotechnology (1992) 10:169-175).

The nucleotide and predicted amino acid sequences of thesignal-CTLA4-IgG gene fusion segments are shown in the Sequence Listingas follows: sigCTLA4-IgGm—SEQ ID NOS: 23 and 24, sigCTLA4-IgG4—SEQ IDNOS: 25 and 26 and sigCTLA4-IgG4m—SEQ ID NOS: 27 and 28.

B. Construction of a CTLA4Ab Fusion Gene

The extracellular domain of CTLA4 is an immunoglobulin superfamilymember and is responsible for binding to its ligands B7-1 and B7-2. Thereplacement of the heavy and light chain variable domains of an antibodymolecule with the extracellular domain of CTLA4 will result in anantibody-like protein which can bind specifically to B7-1, B7-2 andother CTLA4 ligands with high affinity. The construction of such amolecule using human IgG1 antibody heavy and light chains is describedbelow.

1. Construction of the Heavy Chain Gene

The Ig signal sequence was prepared from template plasmid pSP72IgG1 byPCR amplification using oligonucleotide5′CATTCGCTTACCTCGACAAGCTTGAGATCAC AGTTCTCTCTAC-3′ (SEQ ID NO: 13) as theforward PCR primer and oligonucleotide 5′-GGAGTGGACACCTGTGGAGAG-3′ (SEQID NO: 14) as the reverse primer. The forward PCR primer (SEQ ID NO: 13)contains a HindIII restriction site and part of the 5′ untranslatedsegment of the Ig signal domain. The reverse PCR primer (SEQ ID NO: 14)corresponds to the C-terminus of the natural Ig signal peptide.Amplification with these primers resulted in a 208 bp fragment encodingthe entire Ig signal sequence.

The CTLA4 extracellular domain was prepared from plasmid phCTLA4, whichcontained the entire CTLA4 cDNA sequences by PCR amplification usingoligonucleotide 5′-CTCCACAGGTGTCCACTCCGCAATGCACGTGGCCCAGCC-3′ (SEQ IDNO: 15) as the forward PCR primer and oligonucleotide5′GAGGTTGTAAGGACTCACCTGAAA TCTGGGCTCCGTTGC-3′ (SEQ ID NO: 16) as thereverse primer. The forward primer (SEQ ID NO: 15) contained sequenceshomologous to the 5′ end of the CTLA4 extracellular domain and to the 3′end of the Ig signal domain. The reverse primer (SEQ ID NO: 16)contained the 3′ end of the CTLA4 extracellular domain and interveningsequences, including a splice acceptor site. Amplification with theseprimers yielded a 379 bp fragment containing the CTLA4 extracellulardomain.

An intervening sequence DNA fragment derived from the intron between theantibody variable and constant (CH1) domains was prepared by PCRamplification using oligonucleotide5′GCAACGGAGCCCAGATTTCAGGTGAGTCCTTACAACCTC-3′ (SEQ ID NO: 17) as theforward PCR primer and oligonucleotide 5′GGCTAGATATCTCTAGACTATAAATCTCTGGCCATGAAG-3′ (SEQ ID NO: 18) as the reverse PCR primer. Theforward PCR primer (SEQ ID NO: 17) contains intron sequence and iscomplementary to the 3′ end of the extracellular domain of CTLA4 and iscomplimentary to the CTLA4 reverse PCR primer (SEQ ID NO: 16). Thereverse primer (SEQ ID NO: 18) contains intron sequences and anadditional XbaI restriction site. Amplification with these primersyields a 197 bp fragment.

The PCR fragments encoding the Ig signal, CTLA4 extracellular domain andthe intervening sequence were mixed, denatured and renatured to allowhybridization of complementary ends. The strands were filled in and theproduct amplified using forward (SEQ ID NO: 13) and reverse (SEQ ID NO:18) PCR primers. The product was a 764 bp fragment which encoded the Igsignal, the CTLA4 extracellular domain, an intron sequence flanked byHindIII and XbaI restriction sites. This DNA fragment was digested withHindIII and XbaI and ligated to pSP72IgG1, resulting in the CTLA4extracellular domain being linked to a 5′ Ig signal sequence and a 3′antibody CH1, hinge, CH2, and CH3 domains.

The nucleotide and predicted amino acid sequences of the assembledCTLA4-heavy chain are shown in SEQ ID NOS: 29 and 30, respectively.

2. Construction of the Light Chain Gene

The replacement of a human immunoglobulin antibody light chain variabledomain (Hieter, P. A., et al., (1980) Cell 22:197) with the CTLA4extracellular domain proceeded as follows. The Ig signal fragment wasprepared as for the heavy chain replacement, described above. The CTLA4extracellular domain was prepared using a forward PCR primer (SEQ ID NO:15) previously described and oligonucleotide 5′GGCACTAGGTCGACTCTAGAAACTGAGGAAGCAAAGTTTAAATTCTACTCACGTTTAATCTGGGCTCCGTTGC-3′ (SEQ ID NO: 19)as the reverse primer. The reverse primer contained sequences of the 3′end of the CTLA4 extracellular domain, a splice receptor, andintervening sequence DNA containing an XbaI restriction site. The Igsignal fragment and the CTLA4 extracellular domain were joined by mixingthe DNA fragment, denaturing, and renaturing to anneal theircomplementary ends. The strands were filled in and the fragment PCRamplified using forward (SEQ ID NO: 13) and reverse (SEQ ID NO: 19) PCRprimers previously described. The resulting DNA fragment was digestedwith the HindIII and XbaI and ligated to immunoglobulin light chainvector pαLYS17 digested with the same enzymes. The resulting plasmidpCTLA4kappa contains an Ig signal sequence, an intron, the CTLA4extracellular domain, an intron, and the light chain (kappa) constantdomain.

The nucleotide and predicted amino acid sequences of the assembledCTLA4-light chain are shown in SEQ ID NOS: 31 and 32, respectively.

The DNA segments encoding the recombinant heavy and light chains weretransferred to the pEE12 vector or the pNRDSH vector and stable NSO orCHO expression cell lines established as described below. CHO and NSOsupernatants were assayed for the production of CTLA4 light chain andCTLA4 heavy chain fusion proteins by ELISA and binding to B7-1 wasmeasured using CHO/hB7-1 expressing cells and FACS (as described inExample 2). It is also contemplated that the heavy and light chainconstructs of the present invention be expressed in the same vector andhost cells transfected in one step.

C. Expression of CTLA4 Fusion Proteins in CHO and NSO cells

The various CTLA4-immunoglobulin fusion proteins were expressed in CHOcells as follows. Briefly, 5×10⁵ CHO-DG44 cells (subline of CHO-K1,available from ATCC) were transfected with 10 μg of the appropriateexpression plasmid (pNRDSH series) by the calcium phosphate method(described in Molecular Cloning: A Laboratory Manual (1982) Eds.Maniatis, T., Fritsch, E. E., and Sambrook, J. Cold Spring HarborLaboratory) using a commercially available kit (5 Prime to 3′ PrimeInc., Boulder, Colo.) according to the manufacturer's instructions. Thetransfected cells were allowed to recover in nonselective media (alphaMEM medium containing 10% heat inactivate fetal bovine serum (FBS),Gibco/BRL, Gaithersburg, Md.) for two days and then plated in selectivemedia (alpha MEM minus nucleoside medium containing 10% FBS and 550μg/ml G418; Gibco/BRL, Gaithersburg, Md.). Individual subclones wereobtained by dilution cloning in selective media. Culture media wasassayed for the presence of secreted CTLA4-immunoglobulin by a standardELISA designed to detect human IgG.

The various CTLA4-immunoglobulin and CTLA4Ab fusion proteins wereexpressed in NSO cells (Golfre, G. and Milstein C.P. (1981) MethodsEnzymol. 73B: 3-46) as follows. Briefly, 10⁷ NSO cells were transfectedby electroporation (using a BioRad Gene Pulser, Hercules, Calif.) with40 μg of the appropriate expression plasmid (pEE12 series) previouslylinearized by digestion with SalI restriction endonuclease. Thetransfected cells were selected using DMEM media deficient in glutamine(Gibco/BRL, Gaithersberg Md.). Individual subclones were isolated bydilution cloning in selective media. Culture media assayed for thepresence of secreted CTLA4-Ig or CTLA4Ab fusion protein by a standardELISA assay designed to detect human IgG.

As a representative example, transfection of either thepNRDSH/sigCTLA4-IgG4m and pEE12/sigCTLA4-IgG4m expression vector intoCHO or NSO host cells resulted in selected subclones that secretedhCTLA4IgG4m fusion protein into culture supernatants at a concentrationof 75-100 μg/ml.

D. Purification of CTLA4 Fusion Proteins

The CTLA4-Ig and CTLA4Ab fusion proteins are purified from the culturemedium of transfected CHO or NSO cells as follows. Culture medium wasconcentrated 10 fold by ultra filtration (Ultrasette, Filtron TechnologyCorp., Northborough, Mass.) and batch bound overnight to immobilizedprotein A (IPA-300, Repligen Corp., Cambridge, Mass.). The protein-boundresin was poured into a chromatography column, washed with 10 columnvolumes of optimal binding buffer (1.5 M glycine, 3M NaCl, pH 8.9) andthe bound CTLA4-Ig or CTLA4Ab was eluted by the addition of 0.1 M Nacitrate, pH 3.0. Fractions were collected and neutralized with theaddition of 1 M Tris base to pH of 7.0. The Abs_(280 nm) was monitoredfor each fraction and peak fractions were analyzed by SDS-PAGE, followedby Coomassie Blue staining and Western blot analysis using an anti-CTLA4polyclonal antiserum (described in Lindsten, T. et al. (1993) J.Immunol. 151:3489-3499). Fractions containing CTLA4-Ig or CTLA4Ab werepooled and dialyzed against 200 volumes of 0.5×PBS overnight at 4° C.The purified protein was assayed for binding to its ligand (B7-1 and/orB7-2) as described in Example 2.

EXAMPLE 2 Characterization of CTLA4 Fusion Proteins

The ability of the various CTLA4-Ig forms and CTLA4Ab to bind to theircounter receptors B7-1 (Freeman, G. F., et al. (1988) J. Immunol.143:2714-2722) and B7-2 (Freeman, G. F., et al., (1993) Science 262:909-911) was demonstrated using the following assays.

A. Fluorescence Activated Cell Staining (FACS).

Purified preparations of the various recombinant CTLA4 forms were testedfor their ability to bind to transfected COS cell transiently expressinghB7-1 or hB7-2 or transfected CHO cells stably expressing hB7-1 orhB7-2. The recombinant CTLA4 protein (10 μg/ml) was incubated with B7expressing cells (2×10⁶ cells) for 1 hr on ice in FACS wash solution (1%bovine serum albumin in PBS). The cells were washed 3 times with FACSwash solution. The cell bound CTLA4 was detected by reaction withanti-human Ig-FITC (Dako Corporation, Carpintera, Calif.) or proteinA-FITC (Dako) for 30 mintues on ice in the dark. The cells were washedtwice with FACS wash solution and then fixed in 1% paraformaldehyde inPBS. The cells were analyzed for fluorescence intensity using a BectonDickinson (San Jose, Calif.) FACS analyzer. Murine anti-human mAbsreactive with either hB7-1 or hB7-2 served as positive control reagentsfor the hB7-1 and hB7-2 receptor expressing cells. These mAbs weredetected using goat anti-murine IgG-FITC (Dako corporation, Carpintera,Calif.) and analyzed as above. Untransfected COS and CHO cells served asnegative controls for each cell line. The results of this experimentdemonstrated that CTLA4 immunoglobulin fusion proteins bind to CHO cellstransfected to express CTLA4 ligands.

B. Competitive Binding ELISA

The ability of the various recombinant CTLA4 forms to bind to hB7-1 orhB7-2 was assessed in a competitive binding ELISA assay. This assay wasestablished as follows. Purified recombinant hB7-Ig (50 μl at 20 μg/mlin PBS) was bound to a Costar EIA/RIA 96 well microtiter dish (CostarCorp, Cambridge Mass., USA) overnight at room temperature. The wellswere washed three times with 200 μl of PBS and the unbound sites blockedby the addition of 1% BSA in PBS (200 μl/well) for 1 hour at roomtemperature. The wells were washed again as above. BiotinylatedhCTLA4-IgG1 (prepared according to manufacturers instructions (Pierce,Rockford, Ill.) at 10 μg/ml serially diluted in twofold steps to 15.6ng/ml: 50 μl/well) was added to each well and incubated for 2.5 hours atroom temperature. The wells were washed again as above. The boundbiotinylated hCTLA4IgG1 was detected by the addition of 50 μl of a1:2000 dilution of streptavidin-HRP (Pierce Chemical Co., Rockford,Ill.) for 30 minutes at room temperature. The wells were washed as aboveand 50 μl of ABTS (Zymed, Calif.) added and the developing blue colormonitored at 405 nm after 30 min.

The ability of the various forms of CTLA4 to compete with biotinylatedCTLA4-IgG1 was assessed by mixing varying amounts of the competingprotein with a quantity of biotinylated CTLA4-IgG1 shown to benon-saturating (i.e., 70 ng/ml; 1.5 nM) and performing the bindingassays as described above. A reduction in the signal (Abs_(405 nm))expected for biotinylated CTLA4-IgG1 indicated a competition for bindingto plate-bound hB7-1 or hB7-2. A graphic representation of a typicalbinding assay illustrating the competition of biotinylated hCTLA4-IgG1with hCTLA4-IgG1 (itself) or hCTLA4-IgG4m is shown in FIG. 4A forbinding to hB7-1 and FIG. 4B for binding to hB7-2. The competitioncurves show that the mutant IgG4 form competes with hCTLA4-IgG1 forbinding to B7-1 or B7-2 with the same binding kinetics as the unlabeledIgG1 form itself. Accordingly, mutation of the hinge region and CH2domain of IgCγ4 in the CTLA4 fusion protein as described herein does notdetrimentally affect the ligand binding activity of the CTLA4 fusionprotein.

C. SDS-PAGE and Western Blotting

The various CTLA4 forms were analyzed by SDS-PAGE followed by detectionusing Coomassie Blue staining or Western blotting. The CTLA4 proteinswere separated on both reducing and non-reducing SDS-PAGE gels (9, 12,or 15% gels with 5% stacking gel) and stained with Coomassie Blue usingstandard methods. Protein size was estimated from comparison tocommercial size standards (BioRad, Hercules, Calif.). Western blots wereperformed using standard procedures and Immobilon blotting membranes(Millipore, New Bedford, Mass.). The CTLA4 was detected using apolyclonal antisera raised in rabbit immunized with the extracellulardomain of CTLA4 produced in E. coli (described in Lindsten, T. et al.(1993) J. Immunol. 151:3489-3499). The CTLA4 was visualized using[¹²⁵I]-protein A (Dupont NEN, Boston, Mass.) followed by autoradiographyor using protein A-HRP. The results indicated the presence of animmunoreactive band at approximately 50 kD.

D. Measurement of Fc Receptor Binding

The binding of the various CTLA4-Ig forms and CTLA4Ab to Fc receptorswas assessed by using a competitive binding assay as described inAlegre, M.-L., et al., (1992) J. Immunol. 148:3461 3468. Human cell lineU937 was used as a source of the FcRI and FcRII receptors (Looney, R.J., et al., (1986) J. Immunol. 136:1641). U937 cells were grown with 500U/ml IFN-γ to upregulate expression of FcR1. The U937 cells were used ata concentration of 6.25×10⁶ cells/ml. Preparations of unlabeledCTLA4-IgG1, CTLA4-IgG4 and human IgG1 were serially diluted to aconcentration of 2×10⁻¹⁰ M. To each serial dilution, a fixed amount of¹²⁵I-labeled protein (e.g., CTLA4-IgG1, CTLA4-IgG4 or human IgG1) wasadded. The U937 cells were then added to the mixture and incubated forthree hours. The cells were separated from unbound labeled and unlabeledprotein by centrifugation through silicone oil for one minute at14000×g. The tips of the tubes with the pelleted cells were then cut offand analyzed in a gamma counter. Maximal binding of labeled protein toU937 cells was determined in the absence of unlabeled competitorprotein. Percent specific activity represents the percentage of labeledprotein bound in the presence of unlabeled competitor protein relativeto maximal binding. FIG. 5A graphically illustrates the amount oflabeled CTLA4-IgG1 bound to U937 cells (expressed in counts per minute)in the presence of unlabeled CTLA4-IgG1 or CTLA4-IgG4. UnlabeledCTLA4-IgG1 was able to compete with labeled CTLA4-IgG for binding toFcRI on U937 cells (i.e., the amount of bound labeled protein wasreduced), whereas unlabeled CTLA4-IgG4 did not compete for binding. FIG.5B graphically illustrates the percent specific activity of labeledhuman IgG1, CTLA4-IgG1 and CTLA4-IgG4 being competed with themselves(unlabeled). The IC₅₀ for human IgG1 was approximately 7.5×10⁻⁸ M. TheIC₅₀ for CTLA4-IgG1 was approximately 7×10⁻⁸ M. An IC₅₀ for CTLA4-IgG4could not be determined because this protein did not bind to the FcRI.These results demonstrate that use of an IgCγ4 constant region in aCTLA4-Ig fusion protein essentially eliminates the ability of the fusionprotein to bind to Fc receptors.

E. Measurement of Complement Activation

CTLA4-immunoglobulin forms were tested in a ligand-specific assay forcomplement activation. CHO cells expressing hB7-1 on their surface weregrown to confluence in tissue culture dishes. After washing away serumand medium, the cells were exposed to BCECF/AM([2′,7-bis-(carboxyethyl)-5,(6′)-carboxylfluoresceinacetoxymethyl)-ester] Calbiochem, La Jolla, Calif.) a fluorescent dyethat irreversibly loads into the cells. The cells (5×10⁵) were thenincubated with hCTLA4-immunoglobulin fusion proteins or a monoclonalantibody specific for hB7-1 (4B2). Unbound protein was washed away and acomplement source was added and allowed to react with the cells for 30minutes. Complement sources tested included guinea pig complement andhuman serum (as a source of human complement). After incubation with thecomplement source, lysis was measured by monitoring the release of thefluorescent dye from the cells using a fluorometer. Controls includedparallel experiments with hB7-1 negative CHO cells. Identical cultureswere also tested for their ability to bind the hCTLA4 forms undersimilar assay conditions. Additionally, to distinguish a lack of anability to activate complement from a lack of an ability to bind B7-1,an ELISA-type assay of CTLA4 binding to CHO-B7-1 cells was performed asa control (described further below).

The results of typical complement activation assays are shown in FIGS.6A-C. FIG. 6A graphically illustrates guinea pig complement-mediatedlysis of CHO-B7-1 cells by CTLA4-IgG1, CTLA4-IgG4m and the anti-B7-1monoclonal antibody 4B2. hCTLA4-IgG1 reproducibly activated guinea pigcomplement as well or better than the 4B2 mAb. The hCTLA4-IgG4m did notactivate complement in this assay, even at concentration 100-fold higherthan that needed for CTLA4-IgG1. The results were confirmed by repeatingthe work with human serum as the complement source, shown in FIG. 6B.Human complement produced a higher percentage lysis than the guinea pigcomplement, however, otherwise the results were the same, with thehCTLA4-IgG4m exhibiting a markedly reduced ability to activatecomplement in comparison to CTLA4-IgG1. The effect of the CTLA4 fusionproteins on complement activation is specific for the B7-1 ligand, asuntransfected CHO cells were not substrates for complement activation byany of the proteins tested, illustrated in FIG. 6C (using guinea pigcomplement as the complement source).

In order to verify that the hCTLA4-IgG4m form was still able to bind tomembrane bound hB7-1, an experiment was performed by a similar method asfor the complement activation study. Antibody or hCTLA4 forms were boundto washed CHO-B7-1 cells under conditions identical to those used in thecomplement activation studies except that instead of adding complementin the final step, an HRP-conjugated anti-Ig Fc (Calbiochem, La Jolla,Calif.) was used. Bound HRP was detected by washing the cells, addingABTS substrate and measuring absorbence at 405 nm (as described abovefor the competition ELISA assay). The results are shown graphically inFIG. 7. All three B7-1 specific proteins (mAb 4B2. hCTLA4-IgG1 andhCTLA4-IgG4m) bound to the cells. The corresponding experiment usinguntransfected CHO cells showed no binding of the proteins to the cells.The difference in the maximal O.D. signals for the different proteins islikely due to the different affinities of the forms of Fc regions forthe HRP-conjugated secondary antibodies.

F. Inhibition of T Cell Proliferation

The ability of the CTLA4-Ig forms and CTLA4Ab to inhibit theproliferation of T cells in a costimulation proliferation assay wasmeasured. CD4⁺ T cells are prepared from human blood by density gradientcentrifugation on Ficoll-Hypaque (Sigma, St. Louis, Mo.). Monocytes wereremoved by adherence to plastic and the CD4⁺ cells further enriched byremoval of residual monocytes, B cells, NK cells and CD8+ T cells bylysis with complement and mAbs (anti-CD14, antiCD11b, anti-CD20,anti-CD16 and anti-CD8) or by negative selection using the sameimmunomagnetic beads (Advanced Magnetics, Cambridge, Mass.) (asdescribed in Boussioutis, V. A., et al., (1993) J. Exp. Med.178:1758-1763). CD4⁺ T cells (10⁵) were cultured in the presence ofimmobilized anti-CD3 mAb (coated at 1 ug/well, overnight) and CHO cellsexpressing hB7-1 or hB7-2 (2×10⁴) in a microtiter plate with or withoutone of the CTLA4 forms and incubated for 3 days. Thymidine incorporationas a measure of mitogenic activity was assessed after overnightincubation in the presence of [³H] thymidine (Gimmi, C. D., et al.,(1991) Proc. Natl. Acad. Sci USA 88:6575-6579). Inhibition wascalculated as a percent of proliferation in control cultures. The datashow that both the CTLA4IgG1 and CTLA4Ig4m performed well, inhibiting Tcell proliferation to the same extent when used in equivalent amounts,i.e. the two compounds were indistinguishable in potentcy.

G. Pharmacokinetic Studies

The effect of mutating the IgG4 heavy chain, as described herein, on thepharmacokinetics of a CTLA4Ig in rats was examined. Pharmacokineticswere performed on two CTLA4If differing only in their heavy chainconstant domains, where one form contained the wild type human IgG1Ig(referred to as hCTLA4IgG1) and the second antibody contained themutated version of human IgG4 (referred to as hCTLA4IgG4m). TwoSprague-Dawley male rats weighing 0.3-0.4 kg were used for each protein.The CTLA4Ig forms were infused at a dose of 2 mg/kg via a Teflonangiocath which was placed in the marginal ear vein. Two control animalsreceived an infusion of PBS (Ca⁺⁺Mg⁺⁺ free) in the same manner. Bloodsamples were drawn at 0, 15, 30, 60, 90, 360, 480 minutes, 24, 36, 48hours, 7, 14 and 28 days. The concentration of free antibody inheparinized plasma was determined by a standard ELISA. Antibodyclearance rates were determined, α and β t½ values were calculated usingthe P-Fit subroutine of the BIOSOFT Fig-p figure processor/parameterfitter. The results are shown below:

-   -   hCTLA4IgG1        -   α t½=4.2 min        -   β t½=288 min    -   hCTLA4IgG4m        -   α t½=16.6 min        -   β t ½=214.2 min            Both CTLA4IgG1 and CTLA4IgG4m have similar clearance rates,            with a rapid (4-16 min) α phase and a more prolonged            (214-288 min) β phase indicating a serum half life of            approximately 4 hours.

EXAMPLE 3 Preparation of E. coli-Expressed Human CTLA4

A. Intracellular Expression of CTLA4 in E. coli

1. Cloning and Expression of CTLA4 Extracellular Domain

The extracellular domain of CTLA4 was expressed in E. coli after cloninginto expression vector pETCm11a. This vector was derived from expressionvector pET-11a (Novagen Inc., Madison Wis.) by cloning a chloramphenicolresistance gene cassette into the ScaI restriction site within theampicillin resistance gene. The extracellular domain of CTLA4 wasprepared from plasmid phCTLA4 by PCR amplification using oligonucleotide5′GCAGAGAGACAT ATGGCAATGCACGTGGCCCAGCCTGCTGTGG-3′ (SEQ ID NO: 20) asforward primer and oligonucleotide 5′-GCAGAGAGAGGATCCTCAGTCAGTTAGTCAGAATCTGGGCACGGTTCTGG-3′ (SEQID NO: 21) as reverse primer. The forwardPCR primer (SEQ ID NO: 20) contains an NdeI restriction site in whichthe ATG sequence in the NdeI restriction site is followed immediately bythe codon for the first amino acid of mature CTLA4 (Dariavach, P., etal. (1988) Eur. J. Immunol. 18:1901). The reverse PCR primer (SEQ ID NO:21) contains a BamHI restriction site preceded by translation stopcodons in all three reading frames preceded by the last amino acid justprior to the CTLA4 transmembrane domain. PCR amplification with theseprimer yields a 416 bp fragment bounded by NdeI and BamHI restrictionsites which contains DNA sequences encoding the extracellular domain ofCTLA4 preceded by a methionine codon. The PCR product was digested withNdeI plus BamHI and ligated to expression vector pETCm11a digested withthe same restriction enzymes.

The ligated DNA was transfected into E. coli strains BL21, HMS174,RGN714 and RGN715 containing the lambda DE3 helper phage by standardtechniques. Transformants were selected in L-agar containingchloramphenicol at 50 ug/ml. Individual transformants were selected andtested for CTLA4 expression after induction by treatment of cells with0.5 mM IPTG. Whole cell extracts were analyzed on SDS-PAGE gel followedby Coomassie Blue staining and Western blot analysis. The majority ofthe CTLA4 protein in these cells was found in inclusion bodies.

2. Purification of CTLA4 from Inclusion Bodies

Recombinant CTLA4 was recovered from cell pellets by treating the washedcells in lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM PMSF, 5 mM EDTA, 0.5%Triton X-100, and lysozyme at 0.3 mg/ml) followed by sonication. Theinclusion bodies were recovered by centrifugation at 20,000×g andsolubilized by treatment with solubilization buffer (50 mM Tris-HClpH8.0, 8 M urea, 50 mM 2-mercaptoethanol (2-ME)). The solubilization wasassisted by mixing for two hours at room temperature. The solublefraction contained CTLA4. The CTLA4 was purified by chromatography onS-sepharose (Pharmacia, Piscataway, N.J.) as follows. The CTLA4containing supernatant was adjusted to pH 3.4 by the addition of glacialacetic and applied to a S-sepharose column equilibrated in column buffer(100 mM Na-acetate, pH6.5, 8 M urea, 50 mM 2-ME, and 5 mM EDTA). Thecolumn was washed with column buffer and the bound CTLA4 eluted with alinear salt gradient (NaCl, 0 to 1 M) prepared in column buffer. Peakfractions exhibiting high Abs_(280 nm) values were pooled and dialyzedagainst dialysis buffer (100 mM Tris-HCl, pH8.0, 8 M urea, 50 mM, 2-ME,5 mM EDTA). Remaining contaminating proteins were eliminated bychromatography on a Sephacryl S-100 (Pharmacia, Piscataway, N.J.) sizingcolumn. The resulting preparation was greater than 95% pure CTLA4 asestimated by SDS-PAGE followed by Coomassie Blue staining and Westernblot analysis. Since the estimated size of monomeric recombinant CTLA4produced in E. coli was approximately 15 kDa, all steps of thepurification protocol were tested for the presence of a 15 kDa proteinby SDS-PAGE and the presence of CTLA4 verified by Western blotting.

3. Refolding of Denatured CTLA4

The CTLA4 protein purified from inclusion bodies is fully reduced anddenatured and must be properly refolded in a physiological buffer, withintact disulfide bridges. to be in “active” form (i.e., able to bindhB7-1). To avoid solubility problems a step gradient dialysis procedurewas used to remove urea, detergents and reductants. The most successfulrefolding was obtained when the secondary and tertiary protein structurewas encouraged first, by gradient dialysis, removing all urea anddetergent while in the presence of the reductant DTT. Subsequent slowremoval of the DTT appeared to reduce the number of randomintradisulfide bonds. As a control, a sample of CTLA4 was dialyzeddirectly from gel filtration buffer to PBS.

The success of refolding was estimated by immunoprecipitation, 5 μg ofhB7-1-Ig, bound to protein A resin, was used to pull down active CTLA4from a 10 μg aliquot of each refolding trial. Precipitated protein wasrun on a reducing SDS-PAGE, transferred to an Immobilon membrane(Millipore, New Bedford, Mass.) and probed with polyclonal antisera toCTLA4 (antisera 1438, described in Lindsten, T. et al. (1993) J.Immumnol. 151:3489-3499). The relative amount of protein detected at 15kDa was indicative of the success of the refolding process. Refoldingwas also evaluated by assaying CTLA4 binding activity in a competitionELISA as described in Example 2. A successful refolding consisted ofapproximately 5% active protein, or about 2 mg of active protein from a1 L bacterial culture.

B. Preparation of Secreted CTLA4 from E. coli

A secreted form of CTLA4 was prepared from E. coli as follows. Theextracellular domain of CTLA4 was joined to the pelB signal sequence(Lei, S.-P., et al., (1987) J. Bacteriol. 169: 4379-4383) by PCR usingplasmid phCTLA4 as template and oligonucleotide5′GGCACTAGTCATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCGATGGCCGCAGCAATGCACGTGGCCCAGCCTGCTGTGG3′ (SEQ ID NO: 20)as the forward primer and a reverse primer (SEQ ID NO: 21) previouslydescribed. The forward PCR primer 5′-GGCACTAGTCATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGCCCAACCAGCGATGGCCGCAGCAATGCACGTGGCCCAGCCTGCTGTGG-3′ (SEQ ID NO: 22) contains a unique BspHIrestriction site, the complete pelB signal sequence and the 5′ end ofthe extracellular domain of CTLA4. The reverse PCR primer (SEQ ID NO:21) contains a unique BamH1 restriction site preceded by translationalstop codons in all three reading frames preceded by the last amino acidbefore the transmembrane domain of CTLA4. PCR amplification with theseprimers yielded a 480 by fragment bounded by unique BspHI and BamHIrestriction sites encoding the pelB signal sequence joined to the CTLA4extracellular domain.

After PCR amplification, the DNA fragment was digested with BspHI andBamHI and ligated to expression vector pTrc99A (Pharmacia, Piscataway,N.J.) previously digested with NcoI and BamHI. This resulted in aplasmid in which the expression of the pelB-CTLA4 protein was driven bythe pTrc promoter present in the pTrc99A expression vector. E. coli hoststrains transformed with the ligated DNA were selected on L-agarcontaining ampicillin (50 μg/ml) and individual clones isolated. Theexpression of CTLA4 in these strains was induced by the treatment ofexponentially growing cultures with IPTG (0.5 mM) overnight. Extractswere prepared from the culture medium after concentration or by releasefrom periplasm. To prepare periplasmic extracts, cells were incubated in20% sucrose, 10 mM Tris-HCl pH7.5 for 15 minutes at room temperature,collected by centrifugation, and resuspended in 4° C. water and held onice for 10 min. Extracts were assayed for the presence of CTLA4 bySDS-PAGE, Western blotting and competitive B7-1binding ELISA (asdescribed in Example 2). As shown in FIG. 8, soluble CTLA4 prepared fromperiplasmic extracts of E. coli or from the media of these cultures wasable to compete for binding to B7-1 with unlabelled CTLA4Ig. Incontrast, periplasmic extracts from E. coli transfected with the vectoralone or media from these cultures was not able to compete for bindingto B7-1.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-55. (canceled)
 56. A CTLA4-immunoglobulin fusion protein comprising afirst peptide having a CTLA4 activity and a second peptide comprising animmunoglobulin constant region which is modified to reduce at least oneconstant region-mediated biological effector function relative to aCTLA4-IgG1 fusion protein.
 57. A CTLA4-immunoglobulin fusion protein ofclaim 56, wherein the first peptide comprises an extracellular domain ofthe CTLA4 protein.
 58. A CTLA4-immunoglobulin fusion protein of claim57, wherein the first peptide comprises amino acid residues 1-125 of thehuman CTLA4 protein.
 59. A CTLA4-immunoglobulin fusion protein of claim56, wherein the immunoglobulin constant region comprises a hinge region,a CH2 domain and a CH3 domain.
 60. A CTLA4-immunoglobulin fusion proteinof claim 59, wherein the hinge region, the CH2 domain and the CH3 domainare selected from the group consisting of Cγ1, Cγ2, Cγ3 and Cγ4.
 61. ACTLA4-immunoglobulin fusion protein, comprising a first peptide having aCTLA4 activity and a second peptide comprising an immunoglobulinconstant region wherein the immunoglobulin constant region comprises aheavy chain CH1 domain, a hinge region, a CH2 domain and a CH3 domain.62. The peptide of claim 61, wherein the immunoglobulin constant regionis modified to reduce at least one constant region-mediated biologicaleffector function.
 63. The peptide of claim 61, wherein the firstpeptide having a CTLA4 activity and the hinge region of the secondpeptide include at least one cysteine residue available for disulfidebond formation.
 64. The peptide of claim 62, wherein the first peptidehaving a CTLA4 activity and the hinge region of the second peptideinclude at least one cysteine residue available for disulfide bondformation.
 65. A CTLA4-immunoglobulin fusion protein of claim 59,wherein the CH2 domain is modified to reduce biological effectorfunctions.
 66. A CTLA4-immunoglobulin fusion protein of claim 65,wherein the biological effector function is selected from the groupconsisting of complement activation, Fc receptor interaction, andcomplement activation and Fc receptor interaction.
 67. ACTLA4-immunoglobulin fusion protein of claim 66, wherein the CH2 domainis modified by substitution of an amino acid residue located at aposition of an intact immunoglobulin heavy chain selected from the groupconsisting of position 234, position 235 and position
 237. 68. ACTLA4-immunoglobulin fusion protein of claim 67 comprising an amino acidsequence shown in SEQ ID NO:
 24. 69. A CTLA4-immunoglobulin fusionprotein of claim 68 comprising an amino acid sequence shown in SEQ IDNO:
 28. 70. A CTLA4-immunoglobulin light chain fusion protein, whereinthe first peptide comprises a CTLA4 extracellular domain and the secondpeptide comprises an immunoglobulin kappa light chain constant domain.71. An isolated peptide consisting of a CTLA4 extracellular domainproduced by a bacterial host cell, wherein said host cell is transfectedwith an expression vector comprising a nucleotide sequence encoding aCTLA4 extracellular domain.
 72. An isolated peptide consisting of asignal sequence and a CTLA4 extracellular domain produced by a bacterialhost cell, wherein said host cell is transfected with an expressionvector consisting of a nucleotide sequence encoding a signal sequenceand a nucleotide sequence encoding a CTLA4 extracellular domain.
 73. Acomposition suitable for pharmaceutical administration comprising aCTLA4-immunoglobulin fusion protein of claim 56, and a pharmaceuticallyacceptable carrier.
 74. A composition suitable for pharmaceuticaladministration comprising a CTLA4-immunoglobulin fusion protein of claim58, and a pharmaceutically acceptable carrier. 75-91. (canceled)